Methods and Compositions for Increasing Arylsulfatase A Activity in the CNS

ABSTRACT

Provided herein are methods and compositions for treating a subject suffering from a deficiency in arylsulfatase A in the CNS. The methods include systemic administration of a bifunctional fusion antibody comprising an antibody to a human insulin receptor and an arylsulfatase A.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Application No. 61/566,497, filed Dec. 2, 2011, which isincorporated herein in its entirety.

BACKGROUND OF THE INVENTION

Metachromatic leukodystrophy (MLD) is an inherited metabolic diseasecaused by a defect in the lysosomal enzyme arylsulfatase A (ASA), whichfunctions to degrade sulfatides. An insufficient level of ASA causes apathological buildup of 3-O-sulfogalactosyl ceramide (sulfatide), asphingolipid, in, e.g., peripheral tissues, and the central nervoussystem (CNS). Symptoms including neurodegeneration and mentalretardation appear during childhood; and early death can occur due toorgan damage in the brain. Typically, treatment would includeintravenous enzyme replacement therapy with recombinant ASA. However,systemically administered recombinant ASA does not cross the blood brainbarrier (BBB), and therefore has little impact on the effects of thedisease in the CNS.

SUMMARY OF THE INVENTION

Described herein are methods and compositions for treating a subjectsuffering from an arylsulfatase A (“ASA”) deficiency. In certainembodiments, the methods allow delivery of ASA to the CNS bysystemically administering a therapeutically effective amount of abifunctional IgG-ASA fusion protein, where the IgG is an antibody (Ab)that binds an endogenous BBB receptor, such as the human insulinreceptor (HIR). In certain embodiments, the HIR Ab-ASA fusion antibodybinds to the extracellular domain of the insulin receptor and istransported across the blood brain barrier (“BBB”) into the CNS, whileretaining ASA enzyme activity. The HIR Ab binds to the endogenousinsulin receptor on the BBB, and acts as a molecular Trojan horse toferry the ASA into the brain. In certain embodiments, a therapeuticallyeffective systemic dose of a HIR Ab-ASA fusion antibody for systemicadministration is based, in part, on the specific CNS uptakecharacteristics of the fusion antibody from peripheral blood asdescribed herein.

In one aspect provided herein is a method for treating an ASA deficiencyin the central nervous system of a subject in need thereof, comprisingsystemically administering to the subject a therapeutically effectivedose of a fusion antibody having ASA activity. In some embodiments ofthis aspect: (i) the fusion antibody comprises the amino acid sequenceof an immunoglobulin heavy chain, the amino acid sequence of an ASA, andthe amino acid sequence of an immunoglobulin light chain; (ii) thefusion antibody binds to an extracellular domain of the human insulinreceptor and catalyzes hydrolysis of the cerebroside-sulfate groups ofsphingolipids; and (iii) the amino acid sequence of the ASA iscovalently linked to the carboxy terminus of the amino acid sequence ofthe immunoglobulin heavy chain.

In some embodiments at least about 100 ug of ASA enzyme are delivered tothe human brain. In some embodiments, the therapeutically effective doseof the fusion antibody comprises at least about 0.5 mg/Kg of bodyweight. In some embodiments, systemic administration is parenteral,intravenous, subcutaneous, intra-muscular, trans-nasal, intra-arterial,transdermal, or respiratory.

In some embodiments, the brain uptake of the fusion antibody is at least2 fold, 3 fold, 4, fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10fold, 15 fold, 20 fold, 25 fold greater than the brain uptake of acontrol antibody. In some embodiments, the brain volume of distributionof the fusion antibody is at least 2 fold, 3 fold, 4, fold, 5 fold, 6fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 25 fold greaterthan the brain uptake of a control antibody.

In some embodiments, the fusion antibody is a chimeric antibody.

In some embodiments, the immunoglobulin heavy chain of the fusionantibody comprises a CDR1 corresponding to the amino acid sequence ofSEQ ID NO:1 with up to 4 single amino acid mutations, a CDR2corresponding to the amino acid sequence of SEQ ID NO:2 with up to 6single amino acid mutations, or a CDR3 corresponding to the amino acidsequence of SEQ ID NO:3 with up to 3 single amino acid mutations,wherein the single amino acid mutations are substitutions, deletions, orinsertions.

In other embodiments, the immunoglobulin heavy chain of the fusionantibody comprises a CDR1 corresponding to the amino acid sequence ofSEQ ID NO:1 with up to 3 single amino acid mutations, a CDR2corresponding to the amino acid sequence of SEQ ID NO:2 with up to 6single amino acid mutations, and a CDR3 corresponding to the amino acidsequence of SEQ ID NO:3 with up to 3 single amino acid mutations.

In other embodiments, the immunoglobulin heavy chain of the fusionantibody comprises a CDR1 corresponding to the amino acid sequence ofSEQ ID NO:1, a CDR2 corresponding to the amino acid sequence of SEQ IDNO:2, or a CDR3 corresponding to the amino acid sequence of SEQ ID NO:3.

In further embodiments, the complementarity determining region of theimmunoglobulin heavy chain of the fusion antibody comprises a CDR1corresponding to the amino acid sequence of SEQ ID NO:1, a CDR2corresponding to the amino acid sequence of SEQ ID NO:2, and a CDR3corresponding to the amino acid sequence of SEQ ID NO:3.

In some embodiments, the immunoglobulin heavy chain of the fusionantibody comprises a CDR1 corresponding to the amino acid sequence ofSEQ ID NO:1, a CDR2 corresponding to the amino acid sequence of SEQ IDNO:2, and a CDR3 corresponding to the amino acid sequence of SEQ IDNO:3, wherein the amino acid sequences comprise 1, 2, 3, 4, 5, or 6single amino acid mutations.

In some embodiments, the immunoglobulin light chain of the fusionantibody comprises a CDR1 corresponding to the amino acid sequence ofSEQ ID NO:4 with up to 3 single amino acid mutations, a CDR2corresponding to the amino acid sequence of SEQ ID NO:5 with up to 5single amino acid mutations, or a CDR3 corresponding to the amino acidsequence of SEQ ID NO:6 with up to 5 single amino acid mutations,wherein the single amino acid mutations are substitutions, deletions, orinsertions.

In other embodiments, the immunoglobulin light chain of the fusionantibody comprises a CDR1 corresponding to the amino acid sequence ofSEQ ID NO:4 with up to 3 single amino acid mutations, a CDR2corresponding to the amino acid sequence of SEQ ID NO:5 with up to 5single amino acid mutations, and a CDR3 corresponding to the amino acidsequence of SEQ ID NO:6 with up to 5 single amino acid mutations.

In other embodiments, the immunoglobulin light chain of the fusionantibody comprises a CDR1 corresponding to the amino acid sequence ofSEQ ID NO:4, a CDR2 corresponding to the amino acid sequence of SEQ IDNO:5, or a CDR3 corresponding to the amino acid sequence of SEQ ID NO:6.

In further embodiments, the complementarity determining region of theimmunoglobulin light chain of the fusion antibody comprises a CDR1corresponding to the amino acid sequence of SEQ ID NO:4, a CDR2corresponding to the amino acid sequence of SEQ ID NO:5, and a CDR3corresponding to the amino acid sequence of SEQ ID NO:6.

In some embodiments, the immunoglobulin light chain of the fusionantibody comprises a CDR1 corresponding to the amino acid sequence ofSEQ ID NO:4, a CDR2 corresponding to the amino acid sequence of SEQ IDNO:5, or a CDR3 corresponding to the amino acid sequence of SEQ ID NO:6,wherein the amino acid sequences comprise 1, 2, 3, 4, 5, or 6 singleamino acid mutations.

In some embodiments, the immunoglobulin heavy chain of the fusionantibody comprises a CDR1 corresponding to the amino acid sequence ofSEQ ID NO:1, a CDR2 corresponding to the amino acid sequence of SEQ IDNO:2, and a CDR3 corresponding to the amino acid sequence of SEQ IDNO:3; and the immunoglobulin light chain comprises a CDR1 correspondingto the amino acid sequence of SEQ ID NO:4, a CDR2 corresponding to theamino acid sequence of SEQ ID NO:5, and a CDR3 corresponding to theamino acid sequence of SEQ ID NO:6.

In some embodiments, the immunoglobulin heavy chain of the fusionantibody is at least 90% identical to SEQ ID NO:7 and the amino acidsequence of the light chain immunoglobulin is at least 90% identical toSEQ ID NO:8.

In some embodiments, the immunoglobulin heavy chain of the fusionantibody comprises SEQ ID NO:7 and the amino acid sequence of the lightchain immunoglobulin comprises SEQ ID NO:8

In yet further embodiments, the ASA comprises an amino acid sequence atleast 90% (e.g., 95%, or 100%) identical to SEQ ID NO:9.

In other embodiments, the amino acid sequence of the immunoglobulinheavy chain of the fusion antibody at least 90% identical to SEQ IDNO:7; the amino acid sequence of the light chain immunoglobulin is atleast 90% identical to SEQ ID NO:8; and the amino acid sequence of theASA is at least 95% identical to SEQ ID NO:9 or comprises SEQ ID NO:9.

In still other embodiments, the amino acid sequence of theimmunoglobulin heavy chain of the fusion antibody comprises SEQ ID NO:7,the amino acid sequence of the immunoglobulin light chain comprises SEQID NO:8, and the amino acid sequence of the ASA comprises SEQ ID NO:9

In a further aspect provided herein is a method for treating an ASAdeficiency in the central nervous system of a subject in need thereof,comprising systemically administering to the subject a therapeuticallyeffective dose of a fusion antibody having ASA activity, wherein: (i)the fusion antibody comprises: (a) a fusion protein at least 95%identical to SEQ ID NO:10, and (b) an immunoglobulin light chain; and(ii) the fusion antibody binds to an extracellular domain of the humaninsulin receptor and catalyzes hydrolysis of linkages in sulfatidesphingomyelins.

In some embodiments, a fusion protein comprising the amino acidsequences of an immunoglobulin heavy chain and an arylsulfatase Acomprises an amino acid sequence that is at least 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% identical to SEQ ID NO:10.

In yet another aspect provided herein is a method for treating an ASAdeficiency in the central nervous system of a subject in need thereof,comprising systemically administering to the subject a therapeuticallyeffective dose of a fusion antibody having ASA activity, wherein:

(i) the fusion antibody comprises a fusion protein containing the aminoacid sequence of an immunoglobulin heavy chain and an ASA or comprises afusion protein containing the amino acid sequence of an immunoglobulinlight chain and an ASA; the fusion antibody binds to the extracellulardomain of the human insulin receptor; and the fusion antibody catalyzeshydrolysis of linkages in sulfatide sphingomyelin; and (ii) the aminoacid sequence of the ASA is covalently linked to the carboxy terminus ofthe amino acid sequence of the immunoglobulin heavy chain or theimmunoglobulin light chain.

In some embodiments, the ASA deficiency in the central nervous system ismetachromatic leukodystrophy (MLD).

In certain embodiments, provided herein is a fusion antibody comprising:(a) a fusion protein comprising the amino acid sequences of animmunoglobulin heavy chain and an arylsulfatase A, and (b) animmunoglobulin light chain; wherein the fusion antibody crosses theblood brain barrier (BBB) and catalyzes hydrolysis of 2-sulfate groupsof cerebroside sulfate esters and sulfatide sphingolipids.

In some embodiments, the amino acid sequence of the arylsulfatase A iscovalently linked to the carboxy terminus of the amino acid sequence ofthe immunoglobulin heavy chain.

In some embodiments, the fusion antibody is post-translationallymodified by a sulfatase modifying factor type 1 (SUMF1).

In some embodiments, the fusion antibody comprises a formylglycine.

In some embodiments, the fusion protein further comprises a linkerbetween the amino acid sequence of the arylsulfatase A and the carboxyterminus of the amino acid sequence of the immunoglobulin heavy chain.

In some embodiments, the arylsulfatase A specific activity of the fusionantibody is at least about 10 units/mg.

In some embodiments, the ASA retains at least 20% of its activitycompared to its activity as a separate entity. In some embodiments, theASA and the immunoglobulin each retains at least 20% of its activity, ona molar basis, compared to its activity as a separate entity.

In some embodiments, the immunoglobulin heavy chain is an immunoglobulinheavy chain of IgG. In some embodiments, the immunoglobulin heavy chaincomprises a CDR1 corresponding to the amino acid sequence of SEQ IDNO:1, a CDR2 corresponding to the amino acid sequence of SEQ ID NO:2, ora CDR3 corresponding to the amino acid sequence of SEQ ID NO:3.

In some embodiments, the immunoglobulin light chain is an immunoglobulinlight chain of kappa class. In some embodiments, the immunoglobulinlight chain is an immunoglobulin light chain of lambda class. In someembodiments, the immunoglobulin light chain comprises a CDR1corresponding to the amino acid sequence of SEQ ID NO:4, a CDR2corresponding to the amino acid sequence of SEQ ID NO:5, or a CDR3corresponding to the amino acid sequence of SEQ ID NO:6.

In some embodiments, the fusion antibody crosses the BBB by binding anendogenous BBB receptor-mediated transport system. In some embodiments,the fusion antibody crosses the BBB via an endogenous BBB receptorselected from the group consisting of the insulin receptor, transferrinreceptor, leptin receptor, lipoprotein receptor, and the IGF receptor.In some embodiments, the fusion antibody crosses the BBB by binding aninsulin receptor.

In certain embodiments, provided herein is a pharmaceutical compositioncomprising a therapeutically effective amount of a fusion antibodydescribed herein, and a pharmaceutically acceptable excipient.

In some embodiments, provided herein is an isolated polynucleotideencoding the fusion antibody described herein. In some embodiments, theisolated polynucleotide comprises the nucleic acid sequence of SEQ IDNO:14.

In some embodiments, provided herein is a vector comprising the isolatedpolynucleotide described herein. In some embodiments, the vectorprovided herein comprises the nucleic acid sequence of SEQ ID NO:14.

In some embodiments, provided herein is a host cell comprising thevector described herein. In some embodiments, the host cell is a ChineseHamster Ovary (CHO) cell.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings, as follow:

FIG. 1. Schematic depiction of a “molecular trojan horse” strategy inwhich the fusion antibody comprises an antibody to the extracellulardomain of an endogenous BBB receptor (R), which acts as a molecularTrojan horse (TH), and ASA, a lysosomal enzyme (E). Once inside braincells, behind the BBB, the ASA part of the fusion antibody then convertssulfatides (S) to degradable products (P).

FIG. 2. An exemplary HIR Ab-ASA fusion antibody is formed by fusion ofthe amino terminus of the mature ASA to the carboxyl terminus of the CH3region of the heavy chain of the HIR Ab.

FIG. 3. Ethidium bromide stain of agarose gel of human ASA cDNA (leftlane), which was produced by PCR from human liver cDNA, and ASA-specificprimers (Table 2). Middle and right lanes: PhiX174 HaeIII digested DNAstandard, and Lambda HindIII digested DNA standard.

FIG. 4. Genetically engineered tandem vector (TV-HIRMAb-ASA) encoding 4separate and tandem expression cassettes encoding the heavy chain (HC)fusion gene, the light chain (LC) gene, the DHFR gene, and the neo gene.

FIG. 5. Amino acid sequence of an immunoglobulin heavy chain variableregion from an exemplary human insulin receptor antibody directedagainst the extracellular domain of the human insulin receptor. Theunderlined sequences are a signal peptide, CDR1, CDR2, and CDR3,respectively. The heavy chain constant region, taken from human IgG1, isshown in italics.

FIG. 6. Amino acid sequence of an immunoglobulin light chain variableregion from an exemplary human insulin receptor antibody directedagainst the extracellular domain of the human insulin receptor. Theunderlined sequences are a signal peptide, CDR1, CDR2, and CDR3,respectively. The constant region, derived from human kappa light chain,is shown in italics.

FIG. 7. A table showing the CDR1, CDR2, and CDR3 amino acid sequencesfrom a heavy and light chain of an exemplary human insulin receptorantibody directed against the extracellular domain of the human insulinreceptor.

FIG. 8. Amino acid sequence of arylsulfatase A (ASA) (Swiss-ProtP15289), not including the initial 18 amino acid signal peptide (matureASA).

FIG. 9. Amino acid sequence of a fusion of an exemplary human insulinreceptor antibody heavy chain to mature human ASA. The underlinedsequences are, in order, an IgG signal peptide, CDR1, CDR2, CDR3, and apeptide linker (Ser-Ser-Ser) linking the carboxy terminus of the heavychain to the amino terminus of the ASA. Sequence in italic correspondsto the heavy chain constant region, taken from human IgG1. The sequencein bold corresponds to human ASA.

FIG. 10. SDS-PAGE of molecular weight standards, the purified HIRMAb(lane 1), and the purified HIRMAb-ASA fusion protein (lane 2). (A)Reducing SDS-PAGE gel. (B) Non-reducing SDS-PAGE gel.

FIG. 11. Western blot with either anti-human (h) IgG primary antibody(left panel) or anti-human ASA primary antiserum (right panel). Theimmunoreactivity of the HIRMAb-ASA fusion protein is compared to thechimeric HIRMAb. Both the HIRMAb-ASA fusion protein and the HIRMAb haveidentical light chains on the anti-hIgG Western. The HIRMAb-ASA fusionheavy chain reacts with both the anti-hIgG and the anti-human ASAantibody, whereas the HIRMAb heavy chain only reacts with the anti-hIgGantibody.

FIG. 12. Binding of either the chimeric HIRMAb or the HIRMAb-ASA fusionprotein to the HIR extracellular domain (ECD) is saturable. The ED₅₀ ofHIRMAb-ASA binding to the HIR ECD is comparable to the ED₅₀ of thebinding of the chimeric HIRMAb, after normalization for differences inmolecular weight.

FIG. 13. Spectrophotometric assay using para-nitrocatechol sulfate (NCS)as the substrate is used to quantify the ASA specific activity of theHIRMAb-ASA fusion protein at 2 doses of the fusion protein (0.3 and 1.0ug). The assay is linear through 10 minutes of the reaction.

FIG. 14. Plasma concentration of the HIRMAb-ASA fusion protein in theRhesus monkey following intravenous administration, where theconcentration is represented either as a percent of injected dose(ID)/mL (A) or as ng/mL (B).

FIG. 15. Brain scan of the Rhesus monkey at 2 hours after theintravenous injection of the [¹²⁵I]-HIRMAb-ASA fusion protein showsglobal distribution of the fusion protein throughout the primate brainwith higher uptake in gray matter as compared to white matter.

DETAILED DESCRIPTION OF THE INVENTION

The blood brain barrier (BBB) is a severe impediment to the delivery ofsystemically administered ASA (e.g., recombinant ASA) to the centralnervous system. The methods and compositions described herein addressthree factors that are important in delivering a therapeuticallysignificant level of ASA activity across the BBB to the CNS: 1)Modification of an ASA to allow it to cross the BBB via transport on anendogenous BBB transporter; 2) the amount and rate of uptake ofsystemically administered modified ASA into the CNS, via retention ofASA activity following the modification required to produce BBBtransport. Various aspects of the methods and compositions describedherein address these factors, by (1) providing human insulin receptor(HIR) antibody (Ab)-ASA fusion antibodies comprising an ASA (i.e., aprotein having ASA activity) fused, with or without interveningsequence, to an immunoglobulin (heavy chain or light chain) directedagainst the extracellular domain of a human insulin receptor; and (2)establishing therapeutically effective systemic doses of the fusionantibodies based on the uptake in the CNS and the specific activity.

Accordingly, the invention provides compositions and methods fortreating a ASA deficiency in the central nervous system by systemicallyadministering to a subject in need thereof a therapeutically effectivedose of a bifunctional HIR Ab-ASA fusion antibody having ASA activityand selectively binding to the extracellular domain of an endogenous BBBreceptor transporter such as the human insulin receptor.

DEFINITIONS

“Treatment” or “treating” as used herein includes achieving atherapeutic benefit and/or a prophylactic benefit. By therapeuticbenefit is meant eradication or amelioration of the underlying disorderor condition being treated. For example, in an individual with MLD,therapeutic benefit includes partial or complete halting of theprogression of the disorder, or partial or complete reversal of thedisorder. Also, a therapeutic benefit is achieved with the eradicationor amelioration of one or more of the physiological or psychologicalsymptoms associated with the underlying condition such that animprovement is observed in the patient, notwithstanding the fact thatthe patient may still be affected by the condition. A prophylacticbenefit of treatment includes prevention of a condition, retarding theprogress of a condition (e.g., slowing the progression of a lysosomalstorage disorder), or decreasing the likelihood of occurrence of acondition. As used herein, “treating” or “treatment” includesprophylaxis.

As used herein, the term “effective amount” can be an amount, which whenadministered systemically, is sufficient to effect beneficial or desiredresults in the CNS, such as beneficial or desired clinical results, orenhanced cognition, memory, mood, or other desired CNS results. Aneffective amount is also an amount that produces a prophylactic effect,e.g., an amount that delays, reduces, or eliminates the appearance of apathological or undesired condition. Such conditions include, but arenot limited to, mental retardation, hearing loss, and neurodegeneration.An effective amount can be administered in one or more administrations.In terms of treatment, an “effective amount” of a composition of theinvention is an amount that is sufficient to palliate, ameliorate,stabilize, reverse or slow the progression of a disorder, e.g., aneurological disorder. An “effective amount” may be of any of thecompositions of the invention used alone or in conjunction with one ormore agents used to treat a disease or disorder. An “effective amount”of a therapeutic agent within the meaning of the present invention willbe determined by a patient's attending physician or veterinarian. Suchamounts are readily ascertained by one of ordinary skill in the art andwill a therapeutic effect when administered in accordance with thepresent invention. Factors which influence what a therapeuticallyeffective amount will be include, the ASA specific activity of the HIRAb-ASA fusion antibody administered, its absorption profile (e.g., itsrate of uptake into the brain), time elapsed since the initiation of thedisorder, and the age, physical condition, existence of other diseasestates, and nutritional status of the individual being treated.Additionally, other medication the patient may be receiving will affectthe determination of the therapeutically effective amount of thetherapeutic agent to administer.

A “subject” or an “individual,” as used herein, is an animal, forexample, a mammal. In some embodiments a “subject” or an “individual” isa human. In some embodiments, the subject suffers from MLD.

In some embodiments, a pharmacological composition comprising anHIRMAb-ASA fusion antibody is “administered peripherally” or“peripherally administered.” As used herein, these terms refer to anyform of administration of an agent, e.g., a therapeutic agent, to anindividual that is not direct administration to the CNS, i.e., thatbrings the agent in contact with the non-brain side of the blood-brainbarrier. “Peripheral administration,” as used herein, includesintravenous, intra-arterial, subcutaneous, intramuscular,intraperitoneal, transdermal, by inhalation, transbuccal, intranasal,rectal, oral, parenteral, sublingual, or trans-nasal.

A “pharmaceutically acceptable carrier” or “pharmaceutically acceptableexcipient” herein refers to any carrier that does not itself induce theproduction of antibodies harmful to the individual receiving thecomposition. Such carriers are well known to those of ordinary skill inthe art. A thorough discussion of pharmaceutically acceptablecarriers/excipients can be found in Remington's Pharmaceutical Sciences,Gennaro, A R, ed., 20th edition, 2000: Williams and Wilkins PA, USA.Exemplary pharmaceutically acceptable carriers can include salts, forexample, mineral acid salts such as hydrochlorides, hydrobromides,phosphates, sulfates, and the like; and the salts of organic acids suchas acetates, propionates, malonates, benzoates, and the like. Forexample, compositions of the invention may be provided in liquid form,and formulated in saline based aqueous solution of varying pH (5-8),with or without detergents such polysorbate-80 at 0.01-1%, orcarbohydrate additives, such mannitol, sorbitol, or trehalose. Commonlyused buffers include histidine, acetate, phosphate, or citrate.

A “recombinant host cell” or “host cell” refers to a cell that includesan exogenous polynucleotide, regardless of the method used forinsertion, for example, direct uptake, transduction, f-mating, or othermethods known in the art to create recombinant host cells. The exogenouspolynucleotide may be maintained as a nonintegrated vector, for example,a plasmid, or alternatively, may be integrated into the host genome.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues.That is, a description directed to a polypeptide applies equally to adescription of a peptide and a description of a protein, and vice versa.The terms apply to naturally occurring amino acid polymers as well asamino acid polymers in which one or more amino acid residues is anon-naturally occurring amino acid, e.g., an amino acid analog. As usedherein, the terms encompass amino acid chains of any length, includingfull length proteins (i.e., antigens), wherein the amino acid residuesare linked by covalent peptide bonds.

The term “amino acid” refers to naturally occurring and non-naturallyoccurring amino acids, as well as amino acid analogs and amino acidmimetics that function in a manner similar to the naturally occurringamino acids. Naturally encoded amino acids are the 20 common amino acids(alanine, arginine, asparagine, aspartic acid, cysteine, glutamine,glutamic acid, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine, and valine) and pyrolysine and selenocysteine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, such as,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (such as, norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

The term “nucleic acid” refers to deoxyribonucleotides,deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymersthereof in either single- or double-stranded form. Unless specificallylimited, the term encompasses nucleic acids containing known analoguesof natural nucleotides which have similar binding properties as thereference nucleic acid and are metabolized in a manner similar tonaturally occurring nucleotides. Unless specifically limited otherwise,the term also refers to oligonucleotide analogs including PNA(peptidonucleic acid), analogs of DNA used in antisense technology(phosphorothioates, phosphoroamidates, and the like). Unless otherwiseindicated, a particular nucleic acid sequence also implicitlyencompasses conservatively modified variants thereof (including but notlimited to, degenerate codon substitutions) and complementary sequencesas well as the sequence explicitly indicated. Specifically, degeneratecodon substitutions may be achieved by generating sequences in which thethird position of one or more selected (or all) codons is substitutedwith mixed-base and/or deoxyinosine residues (Batzer et al., NucleicAcid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); and Cassol et al. (1992); Rossolini et al., Mol. Cell. Probes8:91-98 (1994)).

The terms “isolated” and “purified” refer to a material that issubstantially or essentially removed from or concentrated in its naturalenvironment. For example, an isolated nucleic acid may be one that isseparated from the nucleic acids that normally flank it or other nucleicacids or components (proteins, lipids, etc. . . . ) in a sample. Inanother example, a polypeptide is purified if it is substantiallyremoved from or concentrated in its natural environment. Methods forpurification and isolation of nucleic acids and proteins are well knownin the art.

The Blood Brain Barrier

In one aspect, the invention provides compositions and methods thatutilize a ASA fused to an immunoglobulin capable of crossing the bloodbrain barrier (BBB) via receptor-mediated transport on an endogenous BBBreceptor/transporter. A preferred endogenous transporter for targetingis the insulin receptor on the BBB. The BBB insulin receptor mediatesthe transport of circulating insulin into the brain, as well as certainpeptidomimetic monoclonal antibodies (MAb) such as the HIRMAb. Otherendogenous transporters that might be targeted with either an endogenousligand or a peptidomimetic MAb include the BBB transferrin receptor, theBBB insulin-like growth factor receptor, the BBB leptin receptor, or theBBB low density lipoprotein receptor. The compositions and methods areuseful in transporting ASA from the peripheral blood and across theblood brain barrier into the CNS. As used herein, the “blood-brainbarrier” refers to the barrier between the peripheral circulation andthe brain and spinal cord which is formed by tight junctions within thebrain capillary endothelial plasma membranes and creates an extremelytight barrier that restricts the transport of molecules into the brain;the BBB is so tight that it is capable of restricting even molecules assmall as urea, molecular weight of 60 Da. The blood-brain barrier withinthe brain, the blood-spinal cord barrier within the spinal cord, and theblood-retinal barrier within the retina, are contiguous capillarybarriers within the central nervous system (CNS), and are collectivelyreferred to as the blood-brain barrier or BBB.

The BBB limits the development of new neurotherapeutics, diagnostics,and research tools for the brain and CNS. Most large moleculetherapeutics such as recombinant proteins, antisense drugs, genemedicines, purified antibodies, or RNA interference (RNAi)-based drugs,do not cross the BBB in pharmacologically significant amounts. While itis generally assumed that small molecule drugs can cross the BBB, infact, <2% of all small molecule drugs are active in the brain owing tothe lack transport across the BBB. A molecule must be lipid soluble andhave a molecular weight less than 400 Daltons (Da) in order to cross theBBB in pharmacologically significant amounts, and the vast majority ofsmall molecules do not have these dual molecular characteristics.Therefore, most potentially therapeutic, diagnostic, or researchmolecules do not cross the BBB in pharmacologically active amounts. Soas to bypass the BBB, invasive transcranial drug delivery strategies areused, such as intracerebro-ventricular (ICV) infusion, intracerebral(IC) administration, and convection enhanced diffusion (CED).Transcranial drug delivery to the brain is expensive, invasive, andlargely ineffective. The ICV route delivers ASA only to the ependymalsurface of the brain, not into brain parenchyma, which is typical fordrugs given by the ICV route. The IC administration of an enzyme such asASA, only provides local delivery, owing to the very low efficiency ofprotein diffusion within the brain. The CED results in preferentialfluid flow through the white matter tracts of brain, which causesdemyelination, and astrogliosis.

The methods described herein offer an alternative to these highlyinvasive and generally unsatisfactory methods for bypassing the BBB,allowing a functional ASA to cross the BBB from the peripheral bloodinto the CNS following systemic administration of an HIRMAb-ASA fusionantibody composition described herein. The methods described hereinexploit the expression of insulin receptors (e.g., human insulinreceptors) on the BBB to shuttle a desired bifunctional HIRMAb-ASAfusion antibody from peripheral blood into the CNS.

Endogenous Receptors

Certain endogenous small molecules in blood, such as glucose or aminoacids, are water soluble, yet are able to penetrate the BBB, owing tocarrier-mediated transport (CMT) on certain BBB carrier systems. Forexample, glucose penetrates the BBB via CMT on the GLUT1 glucosetransporter Amino acids, including therapeutic amino acids such asL-DOPA, penetrate the BBB via CMT on the LAT1 large neutral amino acidtransporter. Similarly, certain endogenous large molecules in blood,such as insulin, transferrin, insulin-like growth factors, leptin, orlow density lipoprotein are able to penetrate the BBB, owing toreceptor-mediated transcytosis (RMT) on certain BBB receptor systems.For example, insulin penetrates the BBB via RMT on the insulin receptor.Transferrin penetrates the BBB via RMT on the transferrin receptor.Insulin-like growth factors may penetrate the BBB via RMT on theinsulin-like growth factor receptor. Leptin may penetrate the BBB viaRMT on the leptin receptor. Low density lipoprotein may penetrate theBBB via transport on the low density lipoprotein receptor.

The BBB has been shown to have specific receptors, including insulinreceptors, that allow the transport from the blood to the brain ofseveral macromolecules. In particular, insulin receptors are suitable astransporters for the HIR Ab-ASA fusion antibodies described herein. TheHIR-ASA fusion antibodies described herein bind to the extracellulardomain (ECD) of the human insulin receptor.

Insulin receptors and their extracellular, insulin binding domain (ECD)have been extensively characterized in the art both structurally andfunctionally. See, e.g., Yip et al (2003), J. Biol. Chem.,278(30):27329-27332; and Whittaker et al. (2005), J Biol Chem,280(22):20932-20936. The amino acid and nucleotide sequences of thehuman insulin receptor can be found under GenBank accession No.NM_(—)000208.

Antibodies that Bind to an Insulin Receptor-Mediated Transport System

One noninvasive approach for the delivery of ASA to the CNS is to fusethe ASA to an antibody that selectively binds to the ECD of the insulinreceptor. Insulin receptors expressed on the BBB can thereby serve as avector for transport of the ASA across the BBB. Certain ECD-specificantibodies may mimic the endogenous ligand and thereby traverse a plasmamembrane barrier via transport on the specific receptor system. Suchinsulin receptor antibodies act as molecular “Trojan horses,” or “TH” asdepicted schematically in FIG. 1. By itself, ASA normally does not crossthe blood-brain barrier (BBB). However, following fusion of the ASA tothe TH, the enzyme is able to cross the BBB, and the brain cellmembrane, by trafficking on the endogenous BBB receptor such as the IR,which is expressed at both the BBB and brain cell membranes in the brain(FIG. 1).

Thus, despite the fact that antibodies and other macromolecules arenormally excluded from the brain, they can be an effective vehicle forthe delivery of molecules into the brain parenchyma if they havespecificity for the extracellular domain of a receptor expressed on theBBB, e.g., the insulin receptor. In certain embodiments, an HIR Ab-ASAfusion antibody binds an exofacial epitope on the human BBB HIR and thisbinding enables the fusion antibody to traverse the BBB via a transportreaction that is mediated by the human BBB insulin receptor.

The term “antibody” describes an immunoglobulin whether natural orpartly or wholly synthetically produced. The term also covers anypolypeptide or protein having a binding domain which is, or ishomologous to, an antigen-binding domain. CDR grafted antibodies arealso contemplated by this term.

“Native antibodies” and “native immunoglobulins” are usuallyheterotetrameric glycoproteins of about 150,000 daltons, composed of twoidentical light (L) chains and two identical heavy (H) chains. Eachlight chain is typically linked to a heavy chain by one covalentdisulfide bond, while the number of disulfide linkages varies among theheavy chains of different immunoglobulin isotypes. Each heavy and lightchain also has regularly spaced intrachain disulfide bridges. Each heavychain has at one end a variable domain (“VH”) followed by a number ofconstant domains (“CH”). Each light chain has a variable domain at oneend (“VL”) and a constant domain (“CL”) at its other end; the constantdomain of the light chain is aligned with the first constant domain ofthe heavy chain, and the light-chain variable domain is aligned with thevariable domain of the heavy chain. Particular amino acid residues arebelieved to form an interface between the light- and heavy-chainvariable domains.

The term “variable domain” refers to protein domains that differextensively in sequence among family members (i.e., among differentisoforms, or in different species). With respect to antibodies, the term“variable domain” refers to the variable domains of antibodies that areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called hypervariable regions both in the light chain andthe heavy chain variable domains. The more highly conserved portions ofvariable domains are called the “framework region” or “FR”. The variabledomains of unmodified heavy and light chains each comprise four FRs(FR1, FR2, FR3 and FR4, respectively), largely adopting a 13-sheetconfiguration, connected by three hypervariable regions, which formloops connecting, and in some cases forming part of, the β-sheetstructure. The hypervariable regions in each chain are held together inclose proximity by the FRs and, with the hypervariable regions from theother chain, contribute to the formation of the antigen-binding site ofantibodies (see Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991), pages 647-669). The constant domains are notinvolved directly in binding an antibody to an antigen, but exhibitvarious effector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region comprises amino acid residues from three“complementarity determining regions” or “CDRs”, which directly bind, ina complementary manner, to an antigen and are known as CDR1, CDR2, andCDR3 respectively.

In the light chain variable domain, the CDRs typically correspond toapproximately residues 24-34 (CDRL1), 50-56 (CDRL2) and 89-97 (CDRL3),and in the heavy chain variable domain the CDRs typically correspond toapproximately residues 31-35 (CDRH1), 50-65 (CDRH2) and 95-102 (CDRH3);Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(1991)) and/or those residues from a “hypervariable loop” (i.e.,residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chainvariable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavychain variable domain; Chothia and Lesk, J. Mol. Biol. 196:901 917(1987)).

As used herein, “variable framework region” or “VFR” refers to frameworkresidues that form a part of the antigen binding pocket or groove and/orthat may contact antigen. In some embodiments, the framework residuesform a loop that is a part of the antigen binding pocket or groove. Theamino acids residues in the loop may or may not contact the antigen. Inan embodiment, the loop amino acids of a VFR are determined byinspection of the three-dimensional structure of an antibody, antibodyheavy chain, or antibody light chain. The three-dimensional structurecan be analyzed for solvent accessible amino acid positions as suchpositions are likely to form a loop and/or provide antigen contact in anantibody variable domain. Some of the solvent accessible positions cantolerate amino acid sequence diversity and others (e.g. structuralpositions) can be less diversified. The three dimensional structure ofthe antibody variable domain can be derived from a crystal structure orprotein modeling. In some embodiments, the VFR comprises, consistessentially of, or consists of amino acid positions corresponding toamino acid positions 71 to 78 of the heavy chain variable domain, thepositions defined according to Kabat et al., 1991. In some embodiments,VFR forms a portion of Framework Region 3 located between CDRH2 andCDRH3. The VFR can form a loop that is well positioned to make contactwith a target antigen or form a part of the antigen binding pocket.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these can be further divided into subclasses(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chainconstant domains (Fc) that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa or (“κ”) and lambda or (“λ”), based on the amino acid sequences oftheir constant domains.

In referring to an antibody or fusion antibody described herein, theterms “selectively bind,” “selectively binding,” “specifically binds,”or “specifically binding” refer to binding to the antibody or fusionantibody to its target antigen for which the dissociation constant (Kd)is about 10⁻⁶ M or lower, i.e., 10⁻⁷, 10⁻⁸, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹, or10⁻¹²M.

The term antibody as used herein will also be understood to mean one ormore fragments of an antibody that retain the ability to specificallybind to an antigen, (see generally, Holliger et al., Nature Biotech. 23(9) 1126-1129 (2005)). Non-limiting examples of such antibodies include(i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CLand CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprisingtwo Fab fragments linked by a disulfide bridge at the hinge region;(iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fvfragment consisting of the VL and VH domains of a single arm of anantibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544 546),which consists of a VH domain; and (vi) an isolated complementaritydetermining region (CDR). Furthermore, although the two domains of theFv fragment, VL and VH, are coded for by separate genes, they can bejoined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the VL and VH regionspair to form monovalent molecules (known as single chain Fv (scFv); seee.g., Bird et al. (1988) Science 242:423 426; and Huston et al. (1988)Proc. Natl. Acad. Sci. USA 85:5879 5883; and Osbourn et al. (1998) Nat.Biotechnol. 16:778). Such single chain antibodies are also intended tobe encompassed within the term antibody. Any VH and VL sequences ofspecific scFv can be linked to human immunoglobulin constant region cDNAor genomic sequences, in order to generate expression vectors encodingcomplete IgG molecules or other isotypes. VH and VL can also be used inthe generation of Fab, Fv or other fragments of immunoglobulins usingeither protein chemistry or recombinant DNA technology. Other forms ofsingle chain antibodies, such as diabodies are also encompassed.

“F(ab′)2” and “Fab′” moieties can be produced by treating immunoglobulin(monoclonal antibody) with a protease such as pepsin and papain, andincludes an antibody fragment generated by digesting immunoglobulin nearthe disulfide bonds existing between the hinge regions in each of thetwo H chains. For example, papain cleaves IgG upstream of the disulfidebonds existing between the hinge regions in each of the two H chains togenerate two homologous antibody fragments in which an L chain composedof VL (L chain variable region) and CL (L chain constant region), and anH chain fragment composed of VH (H chain variable region) and CHγ1 (γ1region in the constant region of H chain) are connected at their Cterminal regions through a disulfide bond. Each of these two homologousantibody fragments is called Fab′. Pepsin also cleaves IgG downstream ofthe disulfide bonds existing between the hinge regions in each of thetwo H chains to generate an antibody fragment slightly larger than thefragment in which the two above-mentioned Fab′ are connected at thehinge region. This antibody fragment is called F(ab′)2.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxyl terminus of the heavy chain CH1 domain including one or morecysteine(s) from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)2 antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and antigen-binding site. This region consists of adimer of one heavy chain and one light chain variable domain in tight,non-covalent association. It is in this configuration that the threehypervariable regions of each variable domain interact to define anantigen-binding site on the surface of the VH-VL dimer Collectively, thesix hypervariable regions confer antigen-binding specificity to theantibody. However, even a single variable domain (or half of an Fvcomprising only three hypervariable regions specific for an antigen) hasthe ability to recognize and bind antigen, although at a lower affinitythan the entire binding site.

“Single-chain Fv” or “sFv” antibody fragments comprise a VH, a VL, orboth a VH and VL domain of an antibody, wherein both domains are presentin a single polypeptide chain. In some embodiments, the Fv polypeptidefurther comprises a polypeptide linker between the VH and VL domainswhich enables the sFv to form the desired structure for antigen binding.For a review of sFv see, e.g., Pluckthun in The Pharmacology ofMonoclonal Antibodies, Vol. 113, Rosenburg and Moore eds.Springer-Verlag, New York, pp. 269 315 (1994).

A “chimeric” antibody includes an antibody derived from a combination ofdifferent mammals. The mammal may be, for example, a rabbit, a mouse, arat, a goat, or a human. The combination of different mammals includescombinations of fragments from human and mouse sources.

In some embodiments, an antibody of the present invention is amonoclonal antibody (MAb), typically a chimeric human-mouse antibodyderived by humanization of a mouse monoclonal antibody. Such antibodiesare obtained from, e.g., transgenic mice that have been “engineered” toproduce specific human antibodies in response to antigenic challenge. Inthis technique, elements of the human heavy and light chain locus areintroduced into strains of mice derived from embryonic stem cell linesthat contain targeted disruptions of the endogenous heavy chain andlight chain loci. The transgenic mice can synthesize human antibodiesspecific for human antigens, and the mice can be used to produce humanantibody-secreting hybridomas.

For use in humans, a HIR Ab is preferred that contains enough humansequence that it is not significantly immunogenic when administered tohumans, e.g., about 80% human and about 20% mouse, or about 85% humanand about 15% mouse, or about 90% human and about 10% mouse, or about95% human and 5% mouse, or greater than about 95% human and less thanabout 5% mouse, or 100% human. A more highly humanized form of the HIRMAb can also be engineered, and the humanized HIR Ab has activitycomparable to the murine HIR Ab and can be used in embodiments of theinvention. See, e.g., U.S. Patent Application Publication Nos.20040101904, filed Nov. 27, 2002 and 20050142141, filed Feb. 17, 2005.Humanized antibodies to the human BBB insulin receptor with sufficienthuman sequences for use in the invention are described in, e.g., Boadoet al. (2007), Biotechnol Bioeng, 96(2):381-391.

In exemplary embodiments, the HIR antibodies or HIR-ASA fusionantibodies derived therefrom contain an immunoglobulin heavy chaincomprising CDRs corresponding to the sequence of at least one of the HCCDRs listed in FIG. 7 (SEQ ID NOs 1-3) or a variant thereof. Forexample, a HC CDR1 corresponding to the amino acid sequence of SEQ IDNO:1 with up to 1, 2, 3, 4, 5, or 6 single amino acid mutations, a HCCDR2 corresponding to the amino acid sequence of SEQ ID NO:2 with up to1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 single amino acid mutations, or a HCCDR3 corresponding to the amino acid sequence of SEQ ID NO:3 with up to1, or 2 single amino acid mutations, where the single amino acidmutations are substitutions, deletions, or insertions.

In other embodiments, the HIR Abs or HIR Ab-ASA fusion Abs contain animmunoglobulin HC the amino acid sequence of which is at least 50%identical (i.e., at least, 55, 60, 65, 70, 75, 80, 85, 90, 95, or anyother percent up to 100% identical) to SEQ ID NO:7 (shown in FIG. 5).

In some embodiments, the HIR Abs or HIR Ab-ASA fusion Abs include animmunoglobulin light chain comprising CDRs corresponding to the sequenceof at least one of the LC CDRs listed in FIG. 7 (SEQ ID NOs: 4-6) or avariant thereof. For example, a LC CDR1 corresponding to the amino acidsequence of SEQ ID NO:4 with up to 1, 2, 3, 4, or 5 single amino acidmutations, a LC CDR2 corresponding to the amino acid sequence of SEQ IDNO:5 with up to 1, 2, 3, or 4 single amino acid mutations, or a LC CDR3corresponding to the amino acid sequence of SEQ ID NO:6 with up to 1, 2,3, 4, or 5 single amino acid mutations.

In other embodiments, the HIR Abs or HIR Ab-ASA fusion Abs contain animmunoglobulin LC the amino acid sequence of which is at least 50%identical (i.e., at least, 55, 60, 65, 70, 75, 80, 85, 90, 95, or anyother percent up to 100% identical) to SEQ ID NO:8 (shown in FIG. 6).

In yet other embodiments, the HIR Abs or HIR Ab-ASA fusion Abs containboth a heavy chain and a light chain corresponding to any of theabove-mentioned HIR heavy chains and HIR light chains.

HIR antibodies used in the invention may be glycosylated ornon-glycosylated. If the antibody is glycosylated, any pattern ofglycosylation that does not significantly affect the function of theantibody may be used. Glycosylation can occur in the pattern typical ofthe cell in which the antibody is made, and may vary from cell type tocell type. For example, the glycosylation pattern of a monoclonalantibody produced by a mouse myeloma cell can be different than theglycosylation pattern of a monoclonal antibody produced by a transfectedChinese hamster ovary (CHO) cell. In some embodiments, the antibody isglycosylated in the pattern produced by a transfected Chinese hamsterovary (CHO) cell.

One of ordinary skill in the art will appreciate that currenttechnologies permit a vast number of sequence variants of candidate HIRAbs or known HIR Abs to be readily generated be (e.g., in vitro) andscreened for binding to a target antigen such as the ECD of the humaninsulin receptor or an isolated epitope thereof. See, e.g., Fukuda etal. (2006) “In vitro evolution of single-chain antibodies using mRNAdisplay,” Nuc. Acid Res., 34(19) (published online) for an example ofμltra high throughput screening of antibody sequence variants. See also,Chen et al. (1999), “In vitro scanning saturation mutagenesis of all thespecificity determining residues in an antibody binding site,” Prot Eng,12(4): 349-356. An insulin receptor ECD can be purified as described in,e.g., Coloma et al. (2000) Pharm Res, 17:266-274, and used to screen forHIR Abs and HIR Ab sequence variants of known HIR Abs.

Accordingly, in some embodiments, a genetically engineered HIR Ab, withthe desired level of human sequences, is fused to an ASA, to produce arecombinant fusion antibody that is a bi-functional molecule. The HIRAb-ASA fusion antibody: (i) binds to an extracellular domain of thehuman insulin receptor; (ii) catalyzes hydrolysis of linkages insulfatides; and (iii) is able to cross the BBB, via transport on the BBBHIR, and retain ASA activity once inside the brain, following peripheraladministration.

Arylsulfatase A (ASA)

Systemic administration (e.g., by intravenous injection) of recombinantASA fails to rescue a deficiency of ASA in the CNS of patients sufferingfrom MLD. ASA does not cross the BBB, and the lack of transport of theenzyme across the BBB prevents it from having a significant therapeuticeffect in the CNS following peripheral administration. However, when theASA is fused to an HIR Ab (e.g., by a covalent linker), this enzyme isnow able to enter the CNS from blood following a non-invasive peripheralroute of administration such as intravenous, intra-arterial,intramuscular, subcutaneous, intraperitoneal, or even oraladministration. Administration of a HIR Ab-ASA fusion antibody enablesdelivery of ASA activity into the brain from peripheral blood. Describedherein is the determination of a systemic dose of the HIR Ab-ASA fusionantibody that is therapeutically effective for treating an ASAdeficiency in the CNS. As described herein, appropriate systemic dosesof an HIR Ab-ASA fusion antibody are established based on a quantitativedetermination of CNS uptake characteristics and enzymatic activity of anHIR Ab-enzyme fusion antibody. Sulfatides are sulfatedgalactosylceramides synthesized primarily in the oligodendrocytes in thecentral nervous system. As used herein, ASA (e.g., the human ASAsequence listed under GenBank Accession No. NP_(—)000478; Swiss-ProtP15289) refers to any naturally occurring or artificial enzyme that cancatalyze the hydrolysis of cerebroside 3-sulfate into cerebroside andsulfate.

ASA is a member of a family of sulfatases that requires a specificpost-translational modification for expression of ASA enzyme activity.The activity of the ASA enzyme is activated following the conversion ofCys-69 (of the intact ASA protein including the signal peptide) to aformylglycine residue by a sulfatase modifying factor type 1 (SUMF1),which is also called the formylglycine generating enzyme (FGE). In someembodiments, the fusion antibody comprising ASA is post-translationallymodified by a sulfatase modifying factor type 1 (SUMF1). In someembodiments, the post-translational modification comprises a cysteine toformylglycine conversion. In some embodiments, the fusion antibodycomprises an ASA that comprises a formylglycine residue.

In some embodiments, ASA has an amino acid sequence that is at least 50%identical (i.e., at least, 55, 60, 65, 70, 75, 80, 85, 90, 95, or anyother percent up to 100% identical) to the amino acid sequence of humanASA, a 507 amino acid protein listed under Swiss-Prot P15289, or a 489amino acid subsequence thereof, which lacks a 18 amino acid signalpeptide, and corresponds to SEQ ID NO:9 (FIG. 8). The structure-functionrelationship of human ASA is well established, as described in, e.g.,von Bulow et al. (2006), “Crystal structure of an enzyme-substratecomplex provides insight into the interaction between humanarylsulfatase a and its substrates during catalysis,” J. Mol. Biol.,305: 26-277, 2001. In particular, residues that are critical to thefunction of ASA include, e.g., Cys-69, Lys-123, Ser-150, His-229, andLys-302.

In some embodiments, ASA has an amino acid sequence at least 50%identical (i.e., at least, 55, 60, 65, 70, 75, 80, 85, 90, 95, or anyother percent up to 100% identical) to SEQ ID NO:9 (shown in FIG. 8).Sequence variants of a canonical ASA sequence such as SEQ ID NO:9 can begenerated, e.g., by random mutagenesis of the entire sequence orspecific subsequences corresponding to particular domains.Alternatively, site directed mutagenesis can be performed reiterativelywhile avoiding mutations to residues known to be critical to ASAfunction such as those given above. Further, in generating multiplevariants of an ASA sequence, mutation tolerance prediction programs canbe used to greatly reduce the number of non-functional sequence variantsthat would be generated by strictly random mutagenesis. Variousprograms) for predicting the effects of amino acid substitutions in aprotein sequence on protein function (e.g., SIFT, PolyPhen, PANTHERPSEC, PMUT, and TopoSNP) are described in, e.g., Henikoff et al. (2006),“Predicting the Effects of Amino Acid Substitutions on ProteinFunction,” Annu. Rev. Genomics Hum. Genet., 7:61-80. ASA sequencevariants can be screened for of ASA activity/retention of ASA activityby p-nitrocatechol sulfate (NCS) spectrophotometric ASA assays known inthe art. One unit of ASA activity is defined as the hydrolysis of 1umole substrate/min at 37C at a defined substrate concentration andreaction conditions. Accordingly, one of ordinary skill in the art willappreciate that a very large number of operable ASA sequence variantscan be obtained by generating and screening extremely diverse“libraries” of ASA sequence variants by methods that are routine in theart, as described above.

Percent sequence identity is determined by conventional methods. See,for example, Altschul et al., Bull. Math. Bio. 48:603 (1986), andHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992).Briefly, two amino acid sequences are aligned to optimize the alignmentscores using a gap opening penalty of 10, a gap extension penalty of 1,and the “BLOSUM62” scoring matrix of Henikoff and Henikoff (ibid.). Thepercent identity is then calculated as: ([Total number of identicalmatches]/[length of the longer sequence plus the number of gapsintroduced into the longer sequence in order to align the twosequences])(100).

Those skilled in the art appreciate that there are many establishedalgorithms available to align two amino acid sequences. The “FASTA”similarity search algorithm of Pearson and Lipman is a suitable proteinalignment method for examining the level of identity shared by an aminoacid sequence disclosed herein and the amino acid sequence of anotherpeptide. The FASTA algorithm is described by Pearson and Lipman, Proc.Nat'l Acad. Sci. USA 85:2444 (1988), and by Pearson, Meth. Enzymol.183:63 (1990). Briefly, FASTA first characterizes sequence similarity byidentifying regions shared by the query sequence (e.g., SEQ ID NO:9 orSEQ ID NO: 16) and a test sequence that have either the highest densityof identities (if the ktup variable is 1) or pairs of identities (ifktup=2), without considering conservative amino acid substitutions,insertions, or deletions. The ten regions with the highest density ofidentities are then rescored by comparing the similarity of all pairedamino acids using an amino acid substitution matrix, and the ends of theregions are “trimmed” to include only those residues that contribute tothe highest score. If there are several regions with scores greater thanthe “cutoff” value (calculated by a predetermined formula based upon thelength of the sequence and the ktup value), then the trimmed initialregions are examined to determine whether the regions can be joined toform an approximate alignment with gaps. Finally, the highest scoringregions of the two amino acid sequences are aligned using a modificationof the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol.Biol. 48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)), whichallows for amino acid insertions and deletions. Illustrative parametersfor FASTA analysis are: ktup=1, gap opening penalty=10, gap extensionpenalty=1, and substitution matrix=BLOSUM62. These parameters can beintroduced into a FASTA program by modifying the scoring matrix file(“SMATRIX”), as explained in Appendix 2 of Pearson, Meth. Enzymol.183:63 (1990).

The present invention also includes proteins having a conservative aminoacid change, compared with an amino acid sequence disclosed herein.Among the common amino acids, for example, a “conservative amino acidsubstitution” is illustrated by a substitution among amino acids withineach of the following groups: (1) glycine, alanine, valine, leucine, andisoleucine, (2) phenylalanine, tyrosine, and tryptophan, (3) serine andthreonine, (4) aspartate and glutamate, (5) glutamine and asparagine,and (6) lysine, arginine and histidine. The BLOSUM62 table is an aminoacid substitution matrix derived from about 2,000 local multiplealignments of protein sequence segments, representing highly conservedregions of more than 500 groups of related proteins (Henikoff andHenikoff, Proc. Nat'l Acad. Sci. USA 89:10915 (1992)). Accordingly, theBLOSUM62 substitution frequencies can be used to define conservativeamino acid substitutions that may be introduced into the amino acidsequences of the present invention. Although it is possible to designamino acid substitutions based solely upon chemical properties (asdiscussed above), the language “conservative amino acid substitution”preferably refers to a substitution represented by a BLOSUM62 value ofgreater than −1. For example, an amino acid substitution is conservativeif the substitution is characterized by a BLOSUM62 value of 0, 1, 2, or3. According to this system, preferred conservative amino acidsubstitutions are characterized by a BLOSUM62 value of at least 1 (e.g.,1, 2 or 3), while more preferred conservative amino acid substitutionsare characterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3).

It also will be understood that amino acid sequences may includeadditional residues, such as additional N- or C-terminal amino acids,and yet still be essentially as set forth in one of the sequencesdisclosed herein, so long as the sequence retains sufficient biologicalprotein activity to be functional in the compositions and methods of theinvention.

Compositions

It has been found that the bifunctional HIR Ab-ASA fusion antibodiesdescribed herein, retain a high proportion of the activity of theirseparate constituent proteins, i.e., binding of the HIR Ab to the IRECD, and the enzymatic activity of ASA. Construction of cDNAs andexpression vectors encoding any of the proteins described herein, aswell as their expression and purification are well within those ofordinary skill in the art, and are described in detail herein in, e.g.,Examples 1-3, and, in Boado et al (2007), Biotechnol Bioeng 96:381-391,U.S. patent application Ser. No. 11/061,956, and U.S. patent applicationSer. No. 11/245,710.

Described herein are bifunctional HIR Ab-ASA fusion antibodiescontaining a HIR Ab, as described herein, capable of crossing the BBBfused to ASA, where the HIR Ab is capable of crossing the blood brainbarrier and the ASA each retain an average of at least about 10, 20, 30,40, 50, 60, 70, 80, 90, 95, 99, or 100% of their activities, compared totheir activities as separate entities. In some embodiments, theinvention provides a HIR Ab-ASA fusion antibody where the HIR Ab and ASAeach retain an average of at least about 50% of their activities,compared to their activities as separate entities. In some embodiments,the invention provides a HIR Ab-ASA fusion antibody where the HIR Ab andASA each retain an average of at least about 60% of their activities,compared to their activities as separate entities. In some embodiments,the invention provides a HIR Ab-ASA fusion antibody where the HIR Ab andASA each retain an average of at least about 70% of their activities,compared to their activities as separate entities. In some embodiments,the invention provides a HIR Ab-ASA fusion antibody where the HIR Ab andASA each retain an average of at least about 80% of their activities,compared to their activities as separate entities. In some embodiments,the invention provides a fusion HIR Ab-ASA fusion antibody where the HIRAb and ASA each retain an average of at least about 90% of theiractivities, compared to their activities as separate entities. In someembodiments, the HIR Ab retains at least about 10, 20, 30, 40, 50, 60,70, 80, 90, 95, 99, or 100% of its activity, compared to its activity asa separate entity, and the ASA retains at least about 10, 20, 30, 40,50, 60, 70, 80, 90, 95, 99, or 100% of its activity, compared to itsactivity as a separate entity. Accordingly, described herein arecompositions containing a bifunctional HIR Ab-ASA fusion antibodycapable of crossing the BBB, where the constituent HIR Ab and ASA eachretain, as part of the fusion antibody, an average of at least about 10,20, 30, 40, 50, 60, 70, 80, 90, 95, 99, or 100% of their activities,i.e., HIR binding and ASA activity, respectively, compared to theiractivities as separate proteins. An HIR Ab ASA fusion antibody refers toa fusion protein comprising any of the HIR antibodies and ASA describedherein.

In the HIR Ab-ASA fusion antibodies described herein, the covalentlinkage between the antibody and the ASA may be to the carboxy or aminoterminal of the HIR antibody and the amino or carboxy terminal of theASA as long as the linkage allows the HIR Ab-ASA fusion antibody to bindto the ECD of the IR and cross the blood brain barrier, and allows theASA to retain a therapeutically useful portion of its activity. Incertain embodiments, the covalent link is between an HC of the antibodyand the ASA or a LC of the antibody and the ASA. Any suitable linkagemay be used, e.g., carboxy terminus of light chain to amino terminus ofASA, carboxy terminus of heavy chain to amino terminus of ASA, aminoterminus of light chain to amino terminus of ASA, amino terminus ofheavy chain to amino terminus of ASA, carboxy terminus of light chain tocarboxy terminus of ASA, carboxy terminus of heavy chain to carboxyterminus of ASA, amino terminus of light chain to carboxy terminus ofASA, or amino terminus of heavy chain to carboxy terminus of ASA. Insome embodiments, the linkage is from the carboxy terminus of the HC tothe amino terminus of the ASA.

The ASA may be fused, or covalently linked, to the targeting antibody(e.g., MAb, HIR-MAb) through a linker. A linkage between terminal aminoacids can be accomplished by an intervening peptide linker sequence thatforms part of the fused amino acid sequence. The peptide sequence linkermay be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 amino acids inlength. In some embodiments, including some preferred embodiments, thepeptide linker is less than 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,4, 3, 2, or 1 amino acids in length. In some embodiments, including somepreferred embodiments, the peptide linker is at least 0, 1, 2, 3, 4, 5,6, 7, 8, 9, 10 amino acids in length. In some embodiments, the ASA isdirectly linked to the targeting antibody, and is therefore 0 aminoacids in length. In some embodiments, there is no linker linking the ASAto the targeting antibody.

In some embodiments, the linker comprises glycine, serine, and/oralanine residues in any combination or order. In some cases, thecombined percentage of glycine, serine, and alanine residues in thelinker is at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%,80%, 90%, or 95% of the total number of residues in the linker. In somepreferred embodiments, the combined percentage of glycine, serine, andalanine residues in the linker is at least 50%, 60%, 70%, 75%, 80%, 90%,or 95% of the total number of residues in the linker. In someembodiments, any number of combinations of amino acids (includingnatural or synthetic amino acids) can be used for the linker. In someembodiments, a three amino acid linker is used. In some embodiments, thelinker has the sequence Ser-Ser-Ser. In some embodiments, a two aminoacid linker comprises glycine, serine, and/or alanine residues in anycombination or order (e.g., Gly-Gly, Ser-Gly, Gly-Ser, Ser-Ser. Ala-Ala,Ser-Ala, or Ala-Ser linker). In some embodiments, a two amino acidlinker consists of one glycine, serine, and/or alanine residue alongwith another amino acid (e.g., Ser-X, where X is any known amino acid).In still other embodiments, the two-amino acid linker consists of anytwo amino acids (e.g., X-X), exept gly, ser, or ala.

As described herein, in some embodiments a linker that is greater thantwo amino acids in length. Such linker may also comprise glycine,serine, and/or alanine residues in any combination or order, asdescribed further herein. In some embodiments, the linker consists ofone glycine, serine, and/or alanine residue along with other amino acids(e.g., Ser-nX, where X is any known amino acid, and n is the number ofamino acids). In still other embodiments, the linker consists of any twoamino acids (e.g., X-X). In some embodiments, said any two amino acidsare Gly, Ser, or Ala, in any combination or order, and within a variablenumber of amino acids intervening between them. In an example of anembodiment, the linker consists of at least one Gly. In an example of anembodiment, the linker consists of at least one Ser. In an example of anembodiment, the linker consists of at least one Ala. In someembodiments, the linker consists of at least 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10 Gly, Ser, and/or Ala residues. In preferred embodiments, thelinker comprises Gly and Ser in repeating sequences, in any combinationor number, such as (Gly₄Ser)₃, or other variations.

A linker for use in the present invention may be designed by using anymethod known in the art. For example, there are multiplepublicly-available programs for determining optimal amino acid linkersin the engineering of fusion proteins. Publicly-available computerprograms (such as the LINKER program) that automatically generate theamino acid sequence of optimal linkers based on the user's input of thesequence of the protein and the desired length of the linker may be usedfor the present methods and compositions. Often, such programs may useobserved trends of naturally-occurring linkers joining proteinsubdomains to predict optimal protein linkers for use in proteinengineering. In some cases, such programs use other methods ofpredicting optimal linkers. Examples of some programs suitable forpredicting a linker for the present invention are described in the art,see, e.g., Xue et al. (2004) Nucleic Acids Res. 32, W562-W565 (WebServer issue providing internet link to LINKER program to assist thedesign of linker sequences for constructing functional fusion proteins);George and Heringa, (2003), Protein Engineering, 15(11):871-879(providing an internet link to a linker program and describing therational design of protein linkers); Argos, (1990), J. Mol. Biol.211:943-958; Arai et al. (2001) Protein Engineering, 14(8):529-532;Crasto and Feng, (2000) Protein Engineering 13(5):309-312.

The peptide linker sequence may include a protease cleavage site,however this is not a requirement for activity of the ASA; indeed, anadvantage of these embodiments of the present invention is that thebifunctional HIR Ab-ASA fusion antibody, without cleavage, is partiallyor fully active both for transport and for activity once across the BBB.FIG. 9 shows an exemplary embodiment of the amino acid sequence of a HIRAb-ASA fusion antibody (SEQ ID NO:10) in which the HC is fused throughits carboxy terminus via a three amino acid “ser-ser-ser” linker to theamino terminus of the ASA. In some embodiments, the fused ASA sequenceis devoid of its 18 amino acid signal peptide, as shown in FIG. 9.

In some embodiments, a HIR Ab-ASA fusion antibody comprises both a HCand a LC. In some embodiments, the HIR Ab-ASA fusion antibody is amonovalent antibody. In other embodiments, the HIR Ab-ASA fusionantibody is a divalent antibody, as described herein in the Examplesection.

The HIR Ab used as part of the HIR Ab-ASA fusion antibody can beglycosylated or nonglycosylated; in some embodiments, the antibody isglycosylated, e.g., in a glycosylation pattern produced by its synthesisin a CHO cell.

As used herein, “activity” includes physiological activity (e.g.,ability to cross the BBB and/or therapeutic activity), binding affinityof the HIR Ab for the IR ECD, or the enzymatic activity of ASA.

Transport of a HIR Ab-ASA fusion antibody across the BBB may be comparedto transport across the BBB of the HIR Ab alone by standard methods. Forexample, pharmacokinetics and brain uptake of the HIR Ab-ASA fusionantibody by a model animal, e.g., a mammal such as a primate, may beused. Similarly, standard models for determining ASA activity may alsobe used to compare the function of the ASA alone and as part of a HIRAb-ASA fusion antibody. See, e.g., Example 4, which demonstrates theenzymatic activity of ASA versus HIR Ab-ASA fusion antibody. Bindingaffinity for the IR ECD can be compared for the HIR Ab-ASA fusionantibody versus the HIR Ab alone. See, e.g., Example 4 herein.

Also included herein are pharmaceutical compositions that contain one ormore HIR Ab-ASA fusion antibodies described herein and apharmaceutically acceptable excipient. A thorough discussion ofpharmaceutically acceptable carriers/excipients can be found inRemington's Pharmaceutical Sciences, Gennaro, A R, ed., 20th edition,2000: Williams and Wilkins PA, USA. Pharmaceutical compositions of theinvention include compositions suitable for administration via anyperipheral route, including intravenous, subcutaneous, intramuscular,intraperitoneal injection; oral, rectal, transbuccal, pulmonary,transdermal, intranasal, or any other suitable route of peripheraladministration.

The compositions of the invention are particular suited for injection,e.g., as a pharmaceutical composition for intravenous, subcutaneous,intramuscular, or intraperitoneal administration. Aqueous compositionsof the present invention comprise an effective amount of a compositionof the present invention, which may be dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium. The phrases“pharmaceutically or pharmacologically acceptable” refer to molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to an animal, e.g., a human,as appropriate. As used herein, “pharmaceutically acceptable carrier”includes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents and thelike. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active ingredient, its use inthe therapeutic compositions is contemplated. Supplementary activeingredients can also be incorporated into the compositions.

Exemplary pharmaceutically acceptable carriers for injectablecompositions can include salts, for example, mineral acid salts such ashydrochlorides, hydrobromides, phosphates, sulfates, and the like; andthe salts of organic acids such as acetates, propionates, malonates,benzoates, and the like. For example, compositions of the invention maybe provided in liquid form, and formulated in saline based aqueoussolution of varying pH (5-8), with or without detergents suchpolysorbate-80 at 0.01-1%, or carbohydrate additives, such mannitol,sorbitol, or trehalose. Commonly used buffers include histidine,acetate, phosphate, or citrate. Under ordinary conditions of storage anduse, these preparations can contain a preservative to prevent the growthof microorganisms. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol; phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate, and gelatin.

For human administration, preparations meet sterility, pyrogenicity,general safety, and purity standards as required by FDA and otherregulatory agency standards. The active compounds will generally beformulated for parenteral administration, e.g., formulated for injectionvia the intravenous, intramuscular, subcutaneous, intralesional, orintraperitoneal routes. The preparation of an aqueous composition thatcontains an active component or ingredient will be known to those ofskill in the art in light of the present disclosure. Typically, suchcompositions can be prepared as injectables, either as liquid solutionsor suspensions; solid forms suitable for use in preparing solutions orsuspensions upon the addition of a liquid prior to injection can also beprepared; and the preparations can also be emulsified.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, methods ofpreparation include vacuum-drying and freeze-drying techniques whichyield a powder of the active ingredient plus any additional desiredingredient from a previously sterile-filtered solution thereof.

Upon formulation, solutions will be systemically administered in amanner compatible with the dosage formulation and in such amount as istherapeutically effective based on the criteria described herein. Theformulations are easily administered in a variety of dosage forms, suchas the type of injectable solutions described above, but drug releasecapsules and the like can also be employed

The appropriate quantity of a pharmaceutical composition to beadministered, the number of treatments, and unit dose will varyaccording to the CNS uptake characteristics of a HIR Ab-ASA fusionantibody as described herein, and according to the subject to betreated, the state of the subject and the effect desired. The personresponsible for administration will, in any event, determine theappropriate dose for the individual subject.

In addition to the compounds formulated for parenteral administration,such as intravenous or intramuscular injection, other alternativemethods of administration of the present invention may also be used,including but not limited to intradermal administration (See U.S. Pat.Nos. 5,997,501; 5,848,991; and 5,527,288), pulmonary administration (SeeU.S. Pat. Nos. 6,361,760; 6,060,069; and 6,041,775), buccaladministration (See U.S. Pat. Nos. 6,375,975; and 6,284,262),transdermal administration (See U.S. Pat. Nos. 6,348,210; and 6,322,808)and transmucosal administration (See U.S. Pat. No. 5,656,284). Suchmethods of administration are well known in the art. One may also useintranasal administration of the present invention, such as with nasalsolutions or sprays, aerosols or inhalants. Nasal solutions are usuallyaqueous solutions designed to be administered to the nasal passages indrops or sprays. Nasal solutions are prepared so that they are similarin many respects to nasal secretions. Thus, the aqueous nasal solutionsusually are isotonic and slightly buffered to maintain a pH of 5.5 to6.5. In addition, antimicrobial preservatives, similar to those used inophthalmic preparations and appropriate drug stabilizers, if required,may be included in the formulation. Various commercial nasalpreparations are known and include, for example, antibiotics andantihistamines and are used for asthma prophylaxis.

Additional formulations, which are suitable for other modes ofadministration, include suppositories and pessaries. A rectal pessary orsuppository may also be used. Suppositories are solid dosage forms ofvarious weights and shapes, usually medicated, for insertion into therectum or the urethra. After insertion, suppositories soften, melt ordissolve in the cavity fluids. For suppositories, traditional bindersand carriers generally include, for example, polyalkylene glycols ortriglycerides; such suppositories may be formed from mixtures containingthe active ingredient in any suitable range, e.g., in the range of 0.5%to 10%, preferably 1%-2%.

Oral formulations include such normally employed excipients as, forexample, pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharine, cellulose, magnesium carbonate and thelike. These compositions take the form of solutions, suspensions,tablets, pills, capsules, sustained release formulations, or powders. Incertain defined embodiments, oral pharmaceutical compositions willcomprise an inert diluent or assimilable edible carrier, or they may beenclosed in a hard or soft shell gelatin capsule, or they may becompressed into tablets, or they may be incorporated directly with thefood of the diet. For oral therapeutic administration, the activecompounds may be incorporated with excipients and used in the form ofingestible tablets, buccal tables, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. Such compositions andpreparations can contain at least 0.1% of active compound. Thepercentage of the compositions and preparations may, of course, bevaried, and may conveniently be between about 2 to about 75% of theweight of the unit, or between about 25-60%. The amount of activecompounds in such therapeutically useful compositions is such that asuitable dosage will be obtained.

The tablets, troches, pills, capsules and the like may also contain thefollowing: a binder, such as gum tragacanth, acacia, cornstarch, orgelatin; excipients, such as dicalcium phosphate; a disintegratingagent, such as corn starch, potato starch, alginic acid and the like; alubricant, such as magnesium stearate; and a sweetening agent, such assucrose, lactose or saccharin may be added or a flavoring agent, such aspeppermint, oil of wintergreen, or cherry flavoring. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, a liquid carrier. Various other materials may be present ascoatings or to otherwise modify the physical form of the dosage unit.For instance, tablets, pills, or capsules may be coated with shellac,sugar or both. A syrup of elixir may contain the active compoundssucrose as a sweetening agent, methylene and propyl parabens aspreservatives, a dye and flavoring, such as cherry or orange flavor. Insome embodiments, an oral pharmaceutical composition may be entericallycoated to protect the active ingredients from the environment of thestomach; enteric coating methods and formulations are well-known in theart.

Methods

Described herein are methods for delivering an effective dose of ASA tothe CNS across the BBB by systemically administering a therapeuticallyeffective amount of a HIR Ab-ASA fusion antibody, as described herein.Suitable systemic doses for delivery of a HIR Ab-ASA fusion antibody isbased on its CNS uptake characteristics and ASA specific activity asdescribed herein. Systemic administration of a HIR Ab-ASA fusionantibody to a subject suffering from an ASA deficiency is an effectiveapproach to the non-invasive delivery of ASA to the CNS.

The amount of a HIR-ASA fusion antibody that is a therapeuticallyeffective systemic dose of a HIR Ab-ASA fusion antibody depends, inpart, on the CNS uptake characteristics of the HIR-ASA fusion antibodyto be administered, as described herein, e.g., the percentage of thesystemically administered dose to be taken up in the CNS.

In some embodiments, 1% (i.e., about 0.3%, 0.4%, 0.48%, 0.6%, 0.74%,0.8%, 0.9%, 1.05, 1.1, 1.2, 1.3%, 1.5%, 2%, 2.5%, 3%, or any % fromabout 0.3% to about 3%) of the systemically administered HIR Ab-ASAfusion antibody is delivered to the brain as a result of its uptake fromperipheral blood across the BBB. In some embodiments, at least 0.5%,(i.e., about 0.3%, 0.4%, 0.48%, 0.6%, 0.74%, 0.8%, 0.9%, 1.05, 1.1, 1.2,1.3%, 1.5%, 2%, 2.5%, 3%, or any % from about 0.3% to about 3%) of thesystemically administered dose of the HIR Ab-ASA fusion antibody isdelivered to the brain within two hours or less, i.e., 1.8, 1.7, 1.5,1.4, 1.3, 1.2, 1.1, 0.9, 0.8, 0.6, 0.5 or any other period from about0.5 to about two hours after systemic administration.

Accordingly, in some embodiments the invention provides methods ofadministering a therapeutically effective amount of a HIR Ab-ASA fusionantibody systemically, such that the amount of the HIR Ab-ASA fusionantibody to cross the BBB provides at least 3 ng of ASA protein/mgprotein in the subject's brain, e.g., 3, 5, 6, 7, 8, 9, 10, 12, 14, 16,18, 20, 30, 40, 50 or any other value from 3 to 50 ng of ASA protein/mgprotein in the subject's brain.

In some embodiments, the total number of units of ASA activity deliveredto a subject's brain is at least, 0.5 milliunits per gram brain, e.g.,at least 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5 or any other totalnumber of ASA units from about 0.5 to 5 milliunits of ASA activitydelivered per gram brain.

In some embodiments, a therapeutically effective systemic dose comprisesat least 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500,2000, 2500 units per brain, or any other systemic dose from about 50 to2500 units of ASA activity per brain.

In other embodiments, a therapeutically effective systemic dose is atleast about 10 units of ASA activity/kg body weight, at least about 10,12, 15, 18, 25, 30, 50, 75, 100, 150, 200, 250, or any other number ofASA units from about 5 to 250 units of ASA activity/kg of body weight.

One of ordinary skill in the art will appreciate that the mass amount ofa therapeutically effective systemic dose of a HIR Ab-ASA fusionantibody will depend, in part, on its ASA specific activity. In someembodiments, the ASA specific activity of a HIR Ab-ASA fusion antibodyis at least 10 U/mg of protein, at least about 10, 12, 14, 16, 18, 20,25, 30, 35, 40, 45, 50, or any other specific activity value from about10 units/mg to about 50 units/mg.

Thus, with due consideration of the specific activity of a HIR Ab-ASAfusion antibody and the body weight of a subject to be treated, asystemic dose of the HIR Ab-ASA fusion antibody can be at least 5 mg,e.g., 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 100,125, or any other value from about 5 mg to about 125 mg of HIR Ab-ASAfusion antibody.

The term “systemic administration” or “peripheral administration,” asused herein, includes any method of administration that is not directadministration into the CNS, i.e., that does not involve physicalpenetration or disruption of the BBB. “Systemic administration”includes, but is not limited to, intravenous, intra-arterialintramuscular, subcutaneous, intraperitoneal, intranasal, transbuccal,transdermal, rectal, transalveolar (inhalation), or oral administration.Any suitable HIR Ab-ASA fusion antibody, as described herein, may beused.

An ASA deficiency as referred to herein includes, one or more conditionsknown as metachromatic leukodystrophy. The ASA deficiency ischaracterized by the buildup of sulfatides that occurs in the body (theheart, liver, brain etc.).

The compositions of the invention, e.g., an HIR Ab-ASA fusion antibody,may be administered as part of a combination therapy. The combinationtherapy involves the administration of a composition of the invention incombination with another therapy for treatment or relief of symptomstypically found in a patient suffering from an ASA deficiency. If thecomposition of the invention is used in combination with another CNSdisorder method or composition, any combination of the composition ofthe invention and the additional method or composition may be used.Thus, for example, if use of a composition of the invention is incombination with another CNS disorder treatment agent, the two may beadministered simultaneously, consecutively, in overlapping durations, insimilar, the same, or different frequencies, etc. In some cases acomposition will be used that contains a composition of the invention incombination with one or more other CNS disorder treatment agents.

In some embodiments, the composition, e.g., an HIR Ab-ASA fusionantibody is co-administered to the patient with another medication,either within the same formulation or as a separate composition. Forexample, the HIR Ab-ASA fusion antibody may be formulated with anotherfusion protein that is also designed to deliver across the humanblood-brain barrier a recombinant protein other than ASA. Further, thefusion HIR Ab-ASA fusion antibody may be formulated in combination withother large or small molecules.

EXAMPLES

The following specific examples are to be construed as merelyillustrative, and not limitative of the remainder of the disclosure inany way whatsoever. Without further elaboration, it is believed that oneskilled in the art can, based on the description herein, utilize thepresent invention to its fullest extent. All publications cited hereinare hereby incorporated by reference in their entirety. Where referenceis made to a URL or other such identifier or address, it is understoodthat such identifiers can change and particular information on theinternet can come and go, but equivalent information can be found bysearching the internet. Reference thereto evidences the availability andpublic dissemination of such information.

Example 1 Expression and functional analysis of HIR Ab-GUSB fusionprotein

The lysosomal enzyme mutated in MPS-VII, also called Sly syndrome, isβ-glucuronidase (GUSB). MPS-VII results in accumulation ofglycosoaminoglycans in the brain. Enzyme replacement therapy (ERT) ofMPS-VII would not likely be effective for treatment of the brain becausethe GUSB enzyme does not cross the BBB. In an effort to re-engineerhuman GUSB to cross the BBB, a HIR Ab-GUSB fusion protein project wasinitiated.

Human GUSB cDNA corresponding to amino acids Met₁-Thr₆₅₁ of the humanGUSB protein (NP_(—)000172), including the 22 amino acid signal peptide,and the 18 amino acid carboxyl terminal propeptide, was cloned byreverse transcription (RT) polymerase chain reaction (PCR) and customoligodexoynucleotides (ODNs). PCR products were resolved in 1% agarosegel electrophoresis, and the expected major single band of ˜2.0 kbcorresponding to the human GUSB cDNA was isolated. The cloned human GUSBwas inserted into a eukaryotic expression plasmid, and this GUSBexpression plasmid was designated pCD-GUSB. The entire expressioncassette of the plasmid was confirmed by bi-directional DNA sequencing.Transfection of COS cells in a 6-well format with the pCD-GSUB resultedin high GUSB enzyme activity in the conditioned medium at 7 days (Table1, Experiment A), which validated the successful engineering of afunctional human GUSB cDNA. The GUSB enzyme activity was determined witha fluorometric assay using 4-methylumbelliferyl beta-L-glucuronide(MUGlcU), which is commercially available. This substrate is hydrolyzedto 4-methylumbelliferone (4-MU) by GUSB, and the 4-MU is detectedfluorometrically with a fluorometer using an emission wavelength of 450nm and an excitation wavelength of 365 nm. A standard curve wasconstructed with known amounts of 4-MU. The assay was performed at 37Cwith 60 min incubations at pH=4.8, and was terminated by the addition ofglycine-carbonate buffer (pH=10.5).

A new pCD-HC-GUSB plasmid expression plasmid was engineered, whichexpresses the fusion protein wherein the carboxyl terminus of the heavychain (HC) of the HIR Ab is fused to the amino terminus of human GUSB,minus the 22 amino acid GUSB signal peptide, and minus the 18 amino acidcarboxyl terminal GUSB propeptide. The GUSB cDNA was cloned by PCR usingthe pCD-GUSB as template. The forward PCR primer introduces “CA”nucleotides to maintain the open reading frame and to introduce aSer-Ser linker between the carboxyl terminus of the CH3 region of theHIR Ab HC and the amino terminus of the GUSB minus the 22 amino acidsignal peptide of the enzyme. The GUSB reverse PCR primer introduces astop codon, “TGA,” immediately after the terminal Thr of the maturehuman GUSB protein. DNA sequencing of the expression cassette of thepCD-HC-GUSB encompassed 4,321 nucleotides (nt), including a 714 ntcytomegalovirus (CMV) promoter, a 9 nt Kozak site (GCCGCCACC), a 3,228nt HC-GUSB fusion protein open reading frame, and a 370 nt bovine growthhormone (BGH) transcription termination sequence. The plasmid encodedfor a 1,075 amino acid protein, comprised of a 19 amino acid IgG signalpeptide, the 443 amino acid HIRMAb HC, a 2 amino acid linker (Ser-Ser),and the 611 amino acid human GUSB minus the enzyme signal peptide andcarboxyl terminal propeptide. The GUSB sequence was 100% identical toLeu²³-Thr⁶³³ of human GUSB (NP_(—)000172). The predicted molecularweight of the heavy chain fusion protein, minus glycosylation, is119,306 Da, with a predicted isoelectric point (pI) of 7.83.

COS cells were plated in 6-well cluster dishes, and were dualtransfected with pCD-LC and pCD-HC-GUSB, where pCD-LC is the expressionplasmid encoding the light chain (LC) of the chimeric HIR Ab.Transfection was performed using Lipofectamine 2000, with a ratio of1:2.5, ug DNA:uL Lipofectamine 2000, and conditioned serum free mediumwas collected at 3 and 7 days. However, there was no specific increasein GUSB enzyme activity following dual transfection of COS cells withthe pCD-HC-GUSB and pCD-LC expression plasmids (Table 1, Experiment B).However, the low GUSB activity in the medium could be attributed to thelow secretion of the HIRMAb-GUSB fusion protein, as the medium IgG wasonly 23±2 ng/mL, as determined by a human IgG-specific ELISA. Therefore,COS cell transfection was scaled up to 10×T500 plates, and theHIRMAb-GUSB fusion protein was purified by protein A affinitychromatography. IgG Western blotting demonstrated the expected increasein size of the fusion protein heavy chain. However, the GUSB enzymeactivity of the HIRMAb-GUSB fusion protein was low at 6.1±0.1 nmol/hr/ugprotein. In contrast, the specific activity of human recombinant GUSB is2,000 nmol/hr/ug protein [Sands et al (1994) Enzyme replacement therapyfor murine mucopolysaccharidosis type VII. J Clin Invest 93, 2324-2331].These results demonstrated the GUSB enzyme activity of the HIR Ab-GUSBfusion protein was >95% lost following fusion of the GUSB to thecarboxyl terminus of the HC of the HIR Ab. The affinity of HIR Ab-GUSBfusion protein binding to the extracellular domain (ECD) of the HIR wasexamined with an ELISA. CHO cells permanently transfected with the HIRECD were grown in serum free media (SFM), and the HIR ECD was purifiedwith a wheat germ agglutinin affinity column. The HIR ECD was plated on96-well dishes and the binding of the HIR Ab, and the HIR Ab-GUSB fusionprotein to the HIR ECD was detected with a biotinylated goat anti-humanIgG (H+L) secondary antibody, followed by avidin and biotinylatedperoxidase. The concentration of protein that gave 50% maximal binding,ED₅₀, was determined with a non-linear regression analysis. The HIRreceptor assay showed there was no decrease in affinity for the HIRfollowing fusion of the 611 amino acid GUSB to the carboxyl terminus ofthe HIRMAb heavy chain. The ED50 of the HIR Ab binding to the HIR ECDwas 0.77±0.10 nM and the ED50 of binding of the HIR Ab-GUSB fusionprotein was 0.81±0.04 nM.

In summary, fusion of the GUSB to the carboxyl terminus of the HIR Ab HCresulted in no loss in affinity of binding of the fusion protein to theHIR. However, the GUSB enzyme activity of the fusion protein wasdecreased by >95%.

In an effort to successfully produce a fusion protein of the HIR Ab andGUSB, a new approach was undertaken, in which the carboxyl terminus ofthe mature human GUSB, including the GUSB signal peptide, was fused tothe amino terminus of the HC of the HIR Ab. This fusion protein wasdesignated GUSB-HIR Ab. The first step was to engineer a new expressionplasmid encoding this new fusion protein, and this plasmid wasdesignated pCD-GUSB-HC. The pCD-GUSB-HC plasmid expresses the fusionprotein wherein the amino terminus of the heavy chain (HC) of theHIRMAb, minus its 19 amino acid signal peptide, is fused to the carboxylterminus of human GUSB, including the 22 amino acid GUSB signal peptide,but minus the 18 amino acid carboxyl terminal GUSB propeptide. ThepCD-GUSB vector was used as template for PCR amplification of the GUSBcDNA expressing a GUSB protein that contained the 22 amino acid GUSBsignal peptide, but lacking the 18 amino acid propeptide at the GUSBcarboxyl terminus. The GUSB 18 amino acid carboxyl terminal propeptidein pCD-GUSB was deleted by site-directed mutagenesis (SDM). The lattercreated an AfeI site on the 3′-flanking region of the Thr⁶³³ residue ofGUSB, and it was designated pCD-GUSB-AfeI. The carboxyl terminalpropeptide was then deleted with AfeI and HindIII (located on the 3′-noncoding region of GUSB). The HIRMAb HC open reading frame, minus the 19amino acid IgG signal peptide and including the HIRMAb HC stop codon,was generated by PCR using the HIRMAb HC cDNA as template. The PCRgenerated HIRMAb HC cDNA was inserted at the AfeI-HindIII sites ofpCD-GUSB-AfeI to form the pCD-GUSB-HC. A Ser-Ser linker between thecarboxyl terminus of GUSB and amino terminus of the HIRMAb HC wasintroduced within the AfeI site by the PCR primer used for the cloningof the HIRMAb HC cDNA. DNA sequencing of the pCD-GUSB-HC expressioncassette showed the plasmid expressed 1,078 amino acid protein,comprised of a 22 amino acid GUSB signal peptide, the 611 amino acidGUSB, a 2 amino acid linker (Ser-Ser), and the 443 amino acid HIRMAb HC.The GUSB sequence was 100% identical to Met¹-Thr⁶³³ of human GUSB(NP_(—)000172).

Dual transfection of COS cells in a 6-well format with the pCD-LC andpCD-GUSB-HC expression plasmids resulted in higher GUSB enzyme activityin the conditioned medium at 7 days, as compared to dual transfectionwith the pCD-LC and pCD-HC-GUSB plasmids (Table 1, Experiment C).However, the GUSB-HIRMAb fusion protein was also secreted poorly by theCOS cells, as the medium human IgG concentration in the 7 dayconditioned medium was only 13±2 ng/mL, as determined by ELISA. COS celltransfection was scaled up to 10×T500 plates, and the GUSB-HIRMAb fusionprotein was purified by protein A affinity chromatography. SDS-PAGEdemonstrated the expected increase in size of the fusion protein heavychain. The GUSB enzyme activity of the purified GUSB-HIRMAb fusionprotein was high at 226±8 nmol/hr/ug protein, which is 37-fold higherthan the specific GUSB enzyme activity of the HIRMAb-GUSB fusionprotein. However, the HIR receptor assay showed there was a markeddecrease in affinity for the HIR following fusion of the GUSB to theamino terminus of the HIRMAb heavy chain, which resulted in a 95%reduction in receptor binding affinity. The ED50 of the HIR Ab bindingto the HIR ECD was 0.25±0.03 nM and the ED50 of binding of the HIRAb-GUSB fusion protein was 4.8±0.4 nM.

In summary, fusion of the GUSB to the amino terminus of the HIR Ab HCresulted in retention of GUSB enzyme activity of the fusion protein, butcaused a 95% reduction in binding of the GUSB-HIR Ab fusion protein tothe HIR. In contrast, fusion of the GUSB to the carboxyl terminus of theHIR Ab HC resulted in no loss in affinity of binding of the HIR Ab-GUSBfusion protein to the HIR. However, the GUSB enzyme activity of thisfusion protein was decreased by >95%. These findings illutstrate theunpredictable nature of the art of fusion of lysosomal enzymes to IgGmolecules in such a way that bi-functionality of the IgG-enzyme fusionprotein is retained, i.e., high affinity binding of the IgG part to thecognate antigen, as well as high enzyme activity.

TABLE 1 GUSB enzyme activity in COS cells following transfection [Mean ±SE (n = 3 dishes per point)] Medium GUSB activity Experiment Treatment(nmol/hour/mL) A Lipofectamine 2000 65 ± 1 pCD-GUSB 6892 ± 631 BLipofectamine 2000 76 ± 3 pCD-HC-GUSB, 72 ± 3 pCD-LC C Lipofectamine2000 162 ± 7  pCD-HC-GUSB, 155 ± 2  pCD-LC pCD-GUSB-HC, 1119 ± 54 pCD-LC

Example 2 Construction of Human HIR Ab Heavy Chain-ASA Fusion ProteinExpression Vector

The lysosomal enzyme mutated in metachromatic leukodystrophy (MLD) isarysulfatase A (ASA). MLD results in accumulation of sulfatides in thebrain, particularly in the myelin sheath. Enzyme replacement therapy ofMLD would likely not be effective for treatment of the brain because theASA enzyme does not cross the BBB. ASA was fused to the HIR Ab in orderto develop a bifunctional molecule capable of both crossing the BBB andexhibiting enzymatic activity. In one embodiment the amino terminus ofthe mature ASA is fused to the carboxyl terminus of each heavy chain ofthe HIR Ab (FIG. 2).

It was unclear whether the enzymatic activity of the ASA would beretained when it was fused to the HIR Ab. The experience with IgG-GUSBfusion proteins described above illustrates the unpredictable nature ofthe art, and the chance that either the IgG part or the lysosomal enzymepart could lose biological activity following construction of theIgG-enzyme fusion protein. The situation with ASA is even more complex,since the ASA enzyme does become catalytically active until there is apost-translational modification of the protein, wherein the Cys residuenear the amino terminus (Cys-515 of SEQ ID NO 10) undergoes apost-translational modification within the endoplasmic reticulum, and itwas not known whether that process would be compromised when ASA wasfused to HIR Ab. ASA is a member of a family of sulfatases, wherein theactivity of the enzyme is activated following the conversion of aspecific Cys residue to a formylglycine residue by a sulfatase modifyingfactor type 1 (SUMF1), also called formylglycine-generating enzyme(FGE), in the endoplasmic reticulum [Takakusaki et al, Coexpression offormylglycine-generating enzyme is essential for synthesis and secretionof functional arylsulfatase A in a mouse model of metachromaticleukodystrophy. Human Gene Ther. 16 (2005) 929-936]. Without thisconversion of the internal cysteine into a formylglycine residue, theenzyme has no activity. If the ASA was fused to the carboxyl terminus ofthe HC of the HIR Ab, e.g. in an effort to retain high affinity bindingof the fusion protein to the HIR, then the IgG heavy chain would foldinto the 3-dimensional structure following translation within the hostcell, followed by folding of the ASA part of the fusion protein. It wasuncertain as to whether the ASA part of the HIR Ab HC-ASA fusion proteinwould fold into a 3-dimensional structure that would be recognized by,and activated by, the ASA-modifying factors in the endoplasmicreticulum, resulting in expression of full ASAenzyme activity in the HIRAb-ASA fusion protein.

The cDNA for the human arylsulfatase A (ARSA) was produced by thepolymerase chain reaction (PCR) using oligodeoxynucleotides (ODN)derived from the nucleotide sequence of the human ASA mRNA (GenBankaccession #NM_(—)000487). The cDNA encoding human ASA, minus its signalpeptide, Arg19-Ala-507, was generated by reverse transcription (RT) PCRusing the ODNs described in Table 2, and commercially available humanliver PolyA+ RNA. The forward (FOR) ODN primer has “CC” on the5′-flanking region to maintain the open reading frame (orf) with the CH3region of human IgG1 in the TV expression vector and to introduce aSer-Ser linker between the human IgG1-CH3 and ASA cDNA. The reverse(REV) ODN is complementary to the end of ASA orf and includes its stopcodon, TGA. RT-PCR was completed and the expected single band of ˜1.5 kbcorresponding to ASA orf cDNA was detected by agarose gelelectrophoresis (FIG. 3, lane 1) and gel purified. Both forward andreverse ODNs are phosphorylated for direct insertion into the expressionvector. The ASA cDNA was inserted into at the HpaI site of a precursorTV, pUTV-1, with T4 DNA ligase to form TV-HIRMAb-ASA, which is outlinedin FIG. 4. The pUTV-1 was linearized with HpaI and digested withalkaline phosphatase to prevent self ligation. The TV-HIRMAb-ASA is atandem vector that encompasses the genes for both the light chain (LC)and heavy chain (HC), respectively, of the HIRMAb-ASA fusion proteinfollowed by the murine dihydrofolate reductase (DHFR) gene. The genesfor the light and heavy chain of the HIRMAb-ASA fusion protein aredriven by the CMV promoter and the orfs are followed by the bovinegrowth hormone (BGH) polyadenylation sequence. The DHFR gene is underthe influence of the SV40 promoter and contains the hepatitis B virus(HBV) polyadenylation termination sequence. The DNA sequence of theTV-HIRMAb-ASA plasmids was confirmed by bi-directional DNA sequencingperformed at MWG Biotech, Inc. (Huntsville, Ala.) using custom ODNssynthesized at Midland (Midland, Tex.). The fusion of the ASA monomer tothe carboxyl terminus of each HC is depicted in FIG. 2. The entireexpression cassette of the plasmid was confirmed by sequencing bothstrands.

TABLE 2 Oligodeoxynucleotide primers used in the RT-PCRcloning of human arylsulfatase A (ASA) minussignal peptide and in the engineering of theHIRMAb-ASA expression vector, derived fromhuman ASA mRNA sequence (GenBank NM_000487).ASA FWD: phosphate-CCCGTCCGCCCAACATCGTGCT (SEQ ID NO. 11)ASA REV: phosphate-TCAGGCATGGGGATCTGGGCAATG (SEQ ID NO. 12)

DNA sequencing of the TV-HIRMAb-ASA plasmid encompassed 9,999nucleotides (nt), which covered the expression cassettes for the LCgene, the HC-ASA gene, and the DHFR gene (FIG. 4). Beginning at the5′-end, the plasmid was comprised of a cytomegalovirus (CMV) promoter, a9 nt full Kozak site, GCCGCCACC (nt 1-9 of SEQ ID NO: 13), a 705 nt openreading frame (orf) for the LC (nt 10-714 of SEQ ID NO: 13), followed bya bovine growth hormone (BGH) polyA sequence, followed by a linkersequence, followed by a tandem CMV promoter, followed by a full Kozaksite (nt 1-9 of SEQ ID NO: 14), followed by a 2,862 nt HIRMAb HC-ASAfusion protein orf (nt 10-2871 of SEQ ID NO: 14), followed by a tandemBGH poly A sequence, followed by the SV40 promoter, followed by a fullKozak site (nt 1-9 of SEQ ID NO: 15), followed by the 564 nt of the DHFRorf (nt 10-573 of SEQ ID NO: 15), followed by the hepatitis B virus polyA sequence (FIG. 4). The TV encoded for a 214 amino acid HIRMAb LC (SEQID NO: 8), which included a 20 amino acid signal peptide; a 953 aminoacid protein fusion protein of the HIRMAb HC and ASA (SEQ ID NO:10). Thefusion protein HC was comprised of a 19 amino acid IgG signal peptide,the 442 amino acid HIRMAb HC, a 3 amino acid linker (Ser-Ser-Ser), andthe 489 amino acid human ASA minus the enzyme signal peptide. Thepredicted molecular weight of the heavy chain fusion protein, minusglycosylation, is 100,637 Da, with a predicted isoelectric point (pI) of6.43. The amino acid sequence of the ASA domain of the HC fusion proteinis 100% identical to the sequence of amino acids 21-509 of human ASA(NP_(—)000478), with the exception of the T391S polymorphism within theASA domain of the fusion protein. This residue is frequently a threonine(T or Thr) residue, but is also known to be a serine (S or Ser) residue.The T391S polymorphism has no effect on the enzyme activity of ASA (S.Regis et al, Contribution of arylsulfatase A mutations located on thesame allele to enzyme activity reduction and metachromaticleukodystrophy severity, Hum. Genet. 110: 351-355, 2002).

Example 3 Stable Transfection of Chinese Hamster Ovary Cells withTV-HIRMAb-ASA

Chinese hamster ovary (CHO) cells were grown in serum free HyQ SFM4-CHOutility medium (HyClone), containing 1×HT supplement (hypoxanthine andthymidine). CHO cells (5×10⁶ viable cells) were electroporated with 5 μgPvuI-linearized TV-HIRMAb-ASA plasmid DNA. The cell-DNA suspension wasthen incubated for 10 min on ice. Cells were electroporated with BioRadpre-set protocol for CHO cells, i.e. square wave with pulse of 15 msecand 160 volts. After electroporation, cells were incubated for 10 min onice. The cell suspension was transferred to 50 ml culture medium andplated at 125 μl per well in 4×96-well plates (10,000 cells per well). Atotal of 10 electroporations and 4,000 wells were performed per study.

Following electroporation (EP), the CHO cells were placed in theincubator at 37 C and 8% CO2. Owing to the presence of the neo gene inthe TV, transfected cell lines were initially selected with G418. TheTV-HIRMAb-ASA also contains the gene for DHFR (FIG. 4), so thetransfected cells were also selected with 20 nM methotrexate (MTX) andHT deficient medium. Once visible colonies were detected at about 21days after EP, the conditioned medium was sampled for human IgG byELISA. Wells with high human IgG signals in the ELISA were transferredfrom the 96-well plate to a 24-well plate with 1 mL of HyQSFM4-CHO-Utility. The 24-well plates were returned to the incubator at37 C and 8% CO2. The following week IgG ELISA was performed on theclones in the 24-well plates. This was repeated through the 6-wellplates to T75 flasks and finally to 60 mL and 125 mL square plasticbottles on an orbital shaker. At this stage, the final MTX concentrationwas 80 nM, and the medium IgG concentration, which was a measure ofHIRMAb-ASA fusion protein in the medium is >10 mg/L at a cell density of10⁶/mL.

Clones selected for dilutional cloning (DC) were removed from theorbital shaker in the incubator and transferred to the sterile hood. Thecells were diluted to 500 mL in F-12K medium with 5% dialyzed fetalbovine serum (d-FBS) and Penicillin/Streptomycin, and the final dilutionis 8 cells per mL, so that 4,000 wells in 40×96-well plates can beplated at a cell density of 1 cell per well (CPW). Once the cellsuspension was prepared, within the sterile hood, a 125 uL aliquot wasdispensed into each well of a 96-well plate using an 8-channel pipettoror a precision pipettor system. The plates were returned to theincubator at 37 C and 8% CO2. The cells diluted to 1 cell/well cannotsurvive without serum. On day 6 or 7, DC plates were removed from theincubator and transferred to the sterile hood where 125 μl of F-12Kmedium with 5% dialyzed fetal bovine serum (d-FBS) was added to eachwell. This selection media now contained 5% d-FBS, 30 nM MTX and 0.25mg/mL Geneticin. On day 21 after the initial 1 CPW plating, aliquotsfrom each of the 4,000 wells were removed for human IgG ELISA, usingrobotics equipment. DC plates were removed from the incubator andtransferred to the sterile hood, where 100 μl of media was removed perwell of the 96-well plate and transferred into a new, sterile sample96-well plate using an 8-channel pipettor or the precision pipettorsystem.

On day 20 after the initial 1 CPW plating, 40×96-well Immunoassay plateswere plated with 100 uL of 1 μg/mL solution of Primary antibody, a mouseanti-human IgG in 0.1M NaHCO3. Plates are incubated overnight in the 4Crefrigerator. The following day, the ELISA plates were washed with1×TBST 5 times, and 100 uL of 1 ug/mL solution of secondary antibody andblocking buffer were added. Plates are washed with 1×TBST 5 times. 100uL of 1 mg/mL of 4-nitrophenyl phosphatedi(2-amino-2-ethyl-1,3-propanediol) salt in 0.1 μM glycine buffer areadded to the 96-well immunoassay plates. Plates were read on amicroplate reader. The assay produced IgG output data for 4,000wells/experiment. The highest producing 24-48 wells were selected forfurther propagation.

The highest producing 24-well plates from the 1 CPW DC were transferredto the sterile hood and gradually subcloned through 6-well dishes, T75flasks, and 125 mL square plastic bottles on an orbital shaker. Duringthis process the serum was reduced to zero, at the final stage ofcentrifugation of the cells and resuspension in SFM.

The above procedures were repeated with a second round of dilutionalcloning, at 0.5-1 cells/well (CPW). At this stage, approximately 40% ofthe wells showed any cell growth, and all wells showing growth alsosecreted human IgG. These results confirmed that on average only 1 cellis plated per well with these procedures, and that the CHO cell lineoriginates from a single cell.

The HIR Ab-ASA fusion protein was secreted to the medium by the stablytransfected CHO cells in high amounts at medium concentrations of 10-20mg/L at a cell density of 1-2 million cells/mL. The high production ofthe HIR Ab-ASA fusion protein by the stably transfected CHO cells wasobserved, even though there was no dual transfection of the host cellwith the fusion protein genes and the gene encoding SUMF1. In cellstransfected with the ASA gene, it was necessary to co-transfect with theSUMF1 co-factor in order to detect secretion of the ASA to the mediumconditioned by the transfected host cell [Takakusaki et al, Coexpressionof formylglycine-generating enzyme is essential for synthesis andsecretion of functional arylsulfatase A in a mouse model ofmetachromatic leukodystrophy. Human Gene Ther. 16 (2005) 929-936]. Anunexpected advantage of engineering ASA and an IgG-ASA fusion protein isthat the host cell secretes the fusion protein without the requirementfor the co-transfection with SUMF1.

The CHO-derived HIRMAb-ASA fusion protein was purified by protein Aaffinity chromatography. The purity of the HIRMAb-ASA fusion protein wasverified by reducing and non-reducing SDS-PAGE as shown in FIGS. 10A and10B, respectively. Only the HC and LC proteins are detected for eitherthe HIRMAb alone or the HIRMAb-ASA fusion protein. The identity of thefusion protein was verified by Western blotting using primary antibodiesto either human IgG (FIG. 11, left panel) or human ASA (FIG. 11, rightpanel). The molecular weight (MW) of the HIRMAb-ASA heavy and lightchains, and the MW of the HIRMAb heavy and light chains are estimated bylinear regression based on the migration of the MW standards. The sizeof the HIRMAb-ASA fusion heavy chain, 119 kDa, is 61 kDa larger than thesize of the heavy chain of the HIRMAb, 58 kDa, owing to the fusion ofthe ASA to the 58 kDa HIRMAb heavy chain. The size of the light chain,25 kDa, is identical for both the HIRMAb-ASA fusion protein and theHIRMAb antibody, as both proteins use the same light chain. Theestimated MW of the hetero-tetrameric HIRMAb-ASA fusion protein shown inFIG. 2 is 288 kDa, based on migration in the SDS-PAGE of the Westernblot.

Example 4 Analysis of HIR Binding and ASA Activity of the Bi-FunctionalIgG-ASA Fusion Protein

The affinity of the fusion protein for the HIR extracellular domain(ECD) was determined with an ELISA. CHO cells permanently transfectedwith the HIR ECD were grown in serum free media (SFM), and the HIR ECDwas purified with a wheat germ agglutinin affinity column, as previouslydescribed in Coloma et al. (2000) Pharm Res, 17:266-274. The HIR ECD wasplated on Nunc-Maxisorb 96 well dishes and the binding of the HIR Ab, orthe HIR Ab-ASA fusion protein, to the HIR ECD was detected with abiotinylated goat anti-human IgG (H+L) secondary antibody, followed byavidin and biotinylated peroxidase (Vector Labs, Burlingame, Calif.).The concentration of either HIR Ab or HIR Ab-ASA fusion protein thatgave 50% maximal binding, ED50, was determined with a non-linearregression analysis. The ED50 of binding to the HIR is 35±9 ng/mL andthe ED50 of binding to the HIR of the HIR Ab-ASA fusion protein is106±33 ng/mL (FIG. 12). The MW of the HIR Ab is 150 kDa, and the MW ofthe HIR Ab-ASA fusion protein is 288 kDa. Therefore, after normalizationfor MW differences, there was comparable binding of either the chimericHIR Ab or the HIR Ab-ASA fusion protein for the HIR ECD with ED50 of0.23±0.06 nM and 0.34±0.11 nM, respectively (FIG. 12). These findingsshow that the affinity of the HIR Ab-ASA fusion protein binding to theHIR is retained, despite fusion of a ASA molecule to the carboxyltermini of both heavy chains of the IgG.

The ASA enzyme activity was determined with a spectrophotometric assayusing p-nitrocatechol sulfate (NCS), which is available from the SigmaCo (St Louis, Mo.). This substrate is hydrolyzed by ASA top-nitrocatechol (NC), which is detected spectrophotometrically at 515nm. A standard curve was constructed with known amounts of NC (Sigma).The assay was performed by incubation at 37C at pH=5.0 for 10 minutes in0.25 M sodium acetate/1 M NaCl/0.25 mM sodium pyrophosphate/0.1% bovineserum albumin The incubation was terminated by the addition of 0.2 mL of1 M NaOH. One unit=1 umol/min. The enzyme activity was linear withrespect to time and mass of fusion protein (FIG. 13), and the averageenzyme activity was 20±1 umol/min/mg protein, or 20 units/mg protein.The ASA enzyme specific activity of recombinant human ASA, using thesame assay, is 60 units/mg protein [Matzner et al (2008): Non-inhibitoryantibodies impede lysosomal storage reduction during enzyme replacementtherapy of a lysosomal storage disease. J. Mol. Med. 86: 433-442].However, following re-engineering of the ASA as an IgG-ASA fusionprotein, the effective MW of the ASA is 144 kDa, whereas the MW of ASAis 60 kDa. Therefore, after normalization for MW differences, theeffective ASA specific activity is 50 units/mg protein, which iscomparable to recombinant ASA. Therefore, fusion of the ASA to thecarboxyl terminus of the HC of the HIR Ab had minimal effect on theenzyme activity of the ASA enzyme, in contrast to the result observedwith the IgG-GUSB fusion protein (Table 1). The high ASA enzyme activityof the CHO-derived HIR Ab-ASA fusion protein is surprising, because ASAis a member of a family of sulfatases that requires a specificpost-translational modification for expression of ASA enzyme activity.The activity of the ASA enzyme is activated following the conversion ofCys-59 to a formylglycine residue by a sulfatase modifying factor type 1(SUMF1), which is also called the formylglycine generating enzyme (FGE).The retention of ASA enzyme activity in the HIRMAb-ASA fusion proteinproduced by the stably transfected CHO cells indicates the ASA enzyme isactivated within the host cell despite fusion to the HIRMAb heavy chain.

Example 5 Amino Acid Linker Joining the ASA and the Targeting Antibody

The mature human ASA is fused to the carboxyl terminus of the HC of theHIR Ab with a 3-amino acid linker, Ser-Ser-Ser (underlined in FIG. 9).Any number of variations of linkers are used as substitutions for theSer-Ser-Ser linker. The 3-amino acid linker may be retained, but theamino acid sequence is changed to alternative amino acids, such asGly-Gly-Gly, or Ser-Gly-Ser, or Ala-Ser-Gly, or any number ofcombinations of the 20 natural amino acids. Or, the linker is reduced toa two, one or zero amino acids. In the case of a zero amino acid linker,the amino terminus of the ASA is fused directly to the carboxyl terminusof the HC of the HIR Ab. Alternatively, the length of the linker isexpanded to 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20 aminoacids. Such linkers are well known in the art, as there are multiplepublicly available programs for determining optimal amino acid linkersin the engineering of fusion proteins. A frequently used linker includesvarious combinations of Gly and Ser in repeating sequences, such as(Gly₄Ser)₃, or other variations

Example 6 HIR Ab-ASA Fusion Protein Uptake and Biological Activity inMLD Fibroblasts

MLD fibroblasts were obtained from the Coriell Institute for MedicalResearch (Camden, N.J.), and grown overnight in DMEM with 10% FBSto >50% confluency in Lab-Tek chamber slide plates. The medium wasaspirated, the wells washed well with PBS, and the cells were treatedwith fresh DMEM with no serum and containing 10 ug/mL of the HIRMAb-ASAfusion protein. Following a 24 hr incubation at 37C, the medium wasaspirated, the wells washed extensively with cold PBS, and the cellswere fixed with 100% cold methanol for 20 min at −20° C. Following a PBSwash, the plates were blocked with 10% donkey serum, and then co-labeledwith 10 ug/mL of a goat anti-ASA antibody, and 10 ug/ml of a mouse MAbto human lysosomal associated membrane protein (LAMP)-1. Negativecontrol antibodies were the same concentrations of either goat or mouseIgG. The secondary antibodies were 5 ug/mL each of Alexa Fluor-488conjugated donkey anti-mouse IgG (green channel) and Alexa Fluor-594conjugated donkey anti-goat IgG (red channel). The washed slides weremounted with Vectashield mounting medium containing4′,6-diamidino-2-phenylindole (DAPI). Confocal microscopy was performedwith a Leica TCS SP2 AOBS inverted fluorescence microscope. Opticalsections (1 um, resolution 300 nm) were obtained sequentially throughthe z-plane of each sample. The LAMP1 immunoreactivity within the cellis detected in the green channel, and the ASA immunoreactivity isdetected in the red channel. There is abundant ASA immunoreactivitywithin the MLD fibroblast indicating the target cell takes up theHIRMAb-ASA fusion protein. The overlap of the ASA and LAMP1immunoreactivity is observed, which means the HIRMAb-ASA fusion proteinis triaged to the lysosomal compartment of the cell. There is noimmunoreactivity in the cells labeled with the isotype controlantibodies, which shows the intracellular LAMP1 and ASA immunoreactivityis specific for the targeted protein.

The intracellular ASA immunoreactivity detected with confocal microscopyrepresents the intact HIRMAb-ASA fusion protein. This was demonstratedwith a Western blotting method. Following incubation of the MLDfibroblasts with the HIRMAb-ASA fusion protein for 4 hours, the mediumwas removed and the cells were washed extensively to removeextracellular fusion protein. The cells were lysed with sodium dodecylsulfate (SDS) sample buffer and the cell extract was analysed with theWestern blot method using a primary antibody against human ASA. Similarto the heavy chain fusion protein shown in FIG. 11 (right panel), theheavy chain of the HIRMAb-ASA fusion protein was detected in the MLDfibroblasts.

Example 7 Receptor-Mediated Delivery of ASA to the Human Brain

Metachromatic leukodystrophy, or MLD, is a lysosomal storage disordercaused by defects in the gene encoding the lysosomal enzyme,arylsulfatase A (ASA). In the absence of ASA, certain sulfoglycolipidsaccumulate in the cells of the brain, including oliogodendrocytes,neurons, and astrocytes (Eckhardt M., The role and metabolism ofsulfatide in the nervous system, Mol Neurobiol, 37: 93-103, 2008). Theaccumulation of the sulfatide glycolipids in the brain leads to theclinical manifestations of MLD, which includes gait disturbances andataxia, spastic quadriplegia, seizures, and eventually death in adecerebrated state. The nucleotide sequence of the ASA mRNA and theamino acid sequence of the human ASA protein is known (C. Stein et al,Cloning and expression of human arylsulfatase A, J. Biol. Chem. 264:1252-1259, 1989). This sequence enables the production of recombinantASA for the enzyme replacement therapy (ERT) of MLD. ASA produced inChinese hamster ovary has a specific activity of 60 units/mg enzyme (U.Matzner et al, Enzyme replacement improves nervous sytem pathology andfunction in a mouse model for metachromatic leukodystrophy, Human Mol.Genet., 14: 1139-1152, 2005). The ASA specific activity is determined bythe p-nitrocatechol sulfate (NCS) spectrophotometric assay (H. Baum etal, The assay of arylsulphatase A and B in human urine, Clin. Chim. Acta4: 453-455, 1959), where 1 unit=1 umol/min (E. Shapira and H. L. Nadler,Purification and some properties of soluble human liver arylsulfatases,Arch. Biochem. Biophys., 170: 179-187, 1975). The problem with ERT ofMLD with recombinant ASA, with respect to treatment of the severeneuropathology of the brain, is that ASA, like other large moleculepharmaceuticals, does not cross the BBB. The administration of a largedose, 40 mg/kg, IV to the MLD mouse does not result in the increase inthe immunoreactive ASA in the mouse brain, and does not result in thedecrease in the sulfatide content in brain in the MLD mouse (U. Matzneret al, Enzyme replacement improves nervous system pathology and functionin a mouse model for metachromatic leukodystrophy, Human Mol. Genet.,14: 1139-1152, 2005). Owing to the lack of transport of ASA across theBBB, it is not possible to increase ASA in brain following the systemicinjection of large doses of the enzyme. Accordingly, ERT in MLD patientswith recombinant ASA was found to have no beneficial effect on the brain(C. i. Dali and A. M. Lund, Intravenous enzyme replacement therapy formetachromatic leukodystrophy (MLD), Abstracts of American CollegeMedical Genetics Annual Meeting, abstract No. 195, 2009. In an attemptto by-pass the BBB by the direct intra-cerebroventricular (ICV) infusionof ASA into the brains of MLD mice, the enzyme was infused into theventricle over 4 weeks (S. Stroobants et al, Intracerebroventricularenzyme infusion corrects central nervous system pathology anddysfunction in a mouse model of metachromatic leukodystrophy, HumanMolec. Genet., 20: 2760-2769, 2011). The tissue half-life of ASA inbrain was <10 minutes following the ICV infusion, whereas the tissuehalf-life of ASA in peripheral tissues following IV administration is 4days (U. Matzner et al, Enzyme replacement improves nervous systempathology and function in a mouse model for metachromaticleukodystrophy, Human Mol. Genet., 14: 1139-1152, 2005). The rapidefflux of ASA from brain following ICV infusion is expected, since anICV injection is like a slow intravenous injection, owing to rapidmovement of the drug from the ventricular compartment to the peripheralvenous circulation. Nevertheless, ASA infusion into the brain wasobserved to correct lysosomal storage disease in the MLD mouse.

Example 8 Receptor-Mediated Delivery of ASA to the Monkey Brain

The treatment of patients with MLD, particularly infants with chronicICV infusion of recombinant ASA, is problematic owing to the requirementof a neurosurgical intervention with placement of intra-cerebralcannulas. However, the more important limitation with the ICV infusiondelivery is the limited penetration of the enzyme into brain parenchymafollowing ICV injection. Owing to the rapid movement of the enzyme fromthe ventricle space to the peripheral blood, there is limited diffusionof the enzyme into brain tissue beyond that which is in contact with theependymal surface of the brain. A preferred approach to the delivery ofASA to the brain of MLD patients is via an intravenous infusion of aform of ASA that is re-engineered to cross the BBB via receptor-mediatedtransport (RMT). The HIRMAb-ASA fusion protein retains high affinitybinding to the human insulin receptor, which enables the sulfatase topenetrate the BBB and enter brain from blood via RMT on the endogenousBBB insulin receptor. The brain uptake of the HIRMAb-sulfatase fusionprotein is 1.1% of injected dose (ID) per 100 grams brain in the Rhesusmonkey, as discussed below. Given this level of brain uptake of thefusion protein, the intravenous administration of 2.5 mg/kg of theIgG-ASA fusion protein, in a 50 kg human, will result in a brainconcentration of 1,375 ug of fusion protein/brain. Since the ASAconstitutes about half of the fusion protein, the brain concentration ofthe ASA enzyme is 687 ug/brain, which is equivalent to 687 ng/gram,since the brain of a human weighs about 1000 grams, and is equivalent to6.9 ng/mg brain protein, since 1 gram of brain contains 100 mg protein.This level of brain uptake of exogenous ASA restores >6% of the normalconcentration of ASA in the human brain, since the concentration ofimmunoreactive ASA in human brain is 100 ng/mg protein (C. Sevin et al,Intracerebral adeno-associated virus-mediated gene transfer in rapidlyprogressive forms of metachromatic leukodystrophy, Human Molec. Genet.,15: 53-64, 2006). Enzyme replacement therapy in patients with lysosomalstorage disorders that produces a cellular enzyme activity of just 1-2%of normal do not develop signs and symptoms of the disease (J. Muenzerand A. Fisher, Advances in the treatment of mucopolysaccharidosis typeI, N. Engl J Med, 350: 1932-1934, 2004). With respect to MLD, there arepatients with ASA pseudo-deficiency, which is about 7% of thepopulation, who are clinically normal but have as little as 3% of thenormal ASA enzyme activity (Penzien J M, et al. (1993) Compoundheterozygosity for metachromatic leukodystrophy and arylsulfatase Apseudodeficiency alleles is not associated with progressive neurologicaldisease. Am J Hum Genet. 52:557-564). These considerations show that aclinically significant ASA enzyme replacement of the human brain ispossible following the intravenous infusion of the HIRMAb-ASA fusionprotein at a systemic dose, 2.5 mg/kg.

The pharmacokinetics and brain uptake of the HIRMAb-ASA fusion proteinin vivo in a living monkey was evaluated with a radiolabeled form of thefusion protein. The HIRMAb-ASA fusion protein was radiolabeled with[¹²⁵I]-Bolton-Hunter reagent to a specific activity of 4.5 uCi/ug and atrichloroacetic acid (TCA) precipitability of 99%. Prior to labeling,the fusion protein was buffer exchanged with 0.01 M sodium acetate/140mM NaCl/pH=5.5/0.001% Tween-80 and an Amicon Ultra-15 centrifugal filterunit. The labeled HIRMAb-ASA fusion protein was purified by gelfiltration with a 1×28 cm column of Sephadex G-25 and an elution bufferof 0.01 M sodium acetate/140 mM NaCl/pH=5.5/0.001% Tween-80. An adultmale Rhesus monkey, 8.2 kg, was investigated at a Contract ResearchOrganization. The animal was injected intravenously (IV) with 2042 uCiof [¹²⁵I]-HIRMAb-ASA fusion protein by bolus injection over 30 secondsin the left femoral vein. The injection dose (ID) of the HIRMAb-ASAfusion protein was 55 ug/kg. The animal was anesthetized withintramuscular ketamine. Following intravenous drug administration,femoral venous plasma was obtained at 2, 5, 15, 30, 60, 90, and 120 minfor determination of total plasma [¹²⁵I] radioactivity (DPM/mL) andplasma radioactivity that is precipitated by 10% cold trichloroaceticacid (TCA). The TCA-precipitable plasma concentration of the fusionprotein is shown in FIG. 14 as either a percent of injected dose (ID)/mLplasma (FIG. 14A) or as ng/mL of fusion protein (FIG. 14B). The percentof total plasma radioactivity that was precipitable by TCA was 98±1%,97±1%, 88±1%, 65±1%, 45±2%, 43±2%, and 42±2%, respectively at 2, 5, 15,30, 60, 90, and 120 min after IV injection. The plasma profile ofTCA-precipitable radioactivity was fit to a 2-exponential equation; theintercepts (A1, A2) and the slopes (k1, k2) of the two exponents ofclearance were used to compute to yield the pharmacokinetics (PK)parameters shown in Table 3. The [¹²⁵I]-HIRMAb-ASA fusion protein israpidly cleared from blood with a mean residence time (MRT) of 59±12minutes, a systemic volume of distribution (Vss) that is 5-fold greaterthe central compartment volume (Vc), and a high rate of systemicclearance (CL), 3.9±0.2 mL/min/kg (Table 3). The plasma area under theconcentration curve (AUC) is shown for the 120 min time period, or thepredicted AUC at steady state, AUCss (Table 3).

TABLE 3 Pharmacokineticparameters of the HIRMAb-ASA fusion proteinparameter units value A1 % ID/mL 0.243 ± 0.034 A2 % ID/mL 0.018 ± 0.003k1 min-1 0.185 ± 0.024 k2 min-1 0.010 ± 0.002 MRT min 59 ± 12 Vc mL/kg46 ± 6  Vss mL/kg 233 ± 39  AUC| ¹²⁰ % ID · min/mL 2.57 ± 0.12 AUCss %ID · min/mL 3.10 ± 0.19 AUCss ug · min/mL 14.2 ± 0.8  CL mL/min/kg 3.9 ±0.2

The uptake of the HIRMAb-ASA fusion protein by brain and peripheralorgans in the primate was measured. The animal was euthanized at 120minutes after fusion protein intravenous injection, and samples of majororgans (heart, liver, spleen, lung, skeletal muscle, and omental fat)were removed, weighed, and processed for determination of radioactivity.The cranium was opened and the brain was removed. Samples of frontalcortical gray matter, frontal cortical white matter, cerebellar graymatter, cerebellar white matter, and choroid plexus were removed forradioactivity determination. The organ uptake of the HIRMAb-ASA fusionprotein, expressed as % of injected dose (ID) per 100 gram wet organweight, in the Rhesus monkey is listed in Table 4 for brain andperipheral organs. The major organs accounting for the removal of theHIRMAb-ASA fusion protein from plasma are liver and spleen (Table 4).The brain uptake of the HIRMAb-ASA fusion protein is 1.1±0.1% ID/100gram brain (Table 4).

TABLE 4 Organ uptake of the HIRMAb-ASA fusion protein in theRhesusmonkey Organ uptake organ (% ID/100 grams) Frontal gray 1.08 ± 0.09Frontal white 0.32 ± 0.10 Cerebellar gray 0.97 ± 0.03 Cerebellar white0.59 ± 0.07 Choroid plexus 2.19 ± 0.68 liver 22.4 ± 1.1  spleen 14.7 ±0.3  lung 3.4 ± 0.2 heart 1.1 ± 0.1 fat 0.33 ± 0.01 Skeletal muscle 0.25± 0.05

The regional uptake by brain of the HIRMAb-ASA fusion protein wasconfirmed by brain scanning at 2 hours after the intravenous injectionof the fusion protein. After euthanasia at 2 hours, the fresh brain wasremoved and cut into coronal slabs, and immediately frozen in liquidnitrogen. Frozen sections (20 um) were cut with a cryostat at −15° C.;the sections were air dried and exposed to X-ray film for up to 7 daysfollowed by x-ray film development. The films were scanned and the imagewas saved in Photoshop, and colorized with NIH Image software. The filmautoradiography of the primate brain shows global distribution of theHIRMAb-ASA fusion protein throughout brain with higher uptake in graymatter as compared to white matter (FIG. 15). Emulsion autoradiographyand light microscopy under dark field and light field illuminationshowed the fusion protein penetrated the BBB and was uniformlydistributed to brain cells within the parenchyma of brain.

The net transport of the HIRMAb-ASA fusion protein through the brainvasculature and into brain parenchyma was confirmed with the capillarydepletion method. The capillary depletion method separates the vasculartissue in brain from the post-vascular compartment (Triguero et al,1990). Based on measurements of the specific activity of braincapillary-specific enzymes, such as γ-glutamyl transpeptidase oralkaline phosphatase, the post-vascular supernatant is >95% depleted ofbrain vasculature (Triguero D, Buciak J, Pardridge W M 1990. Capillarydepletion method for quantification of blood-brain barrier transport ofcirculating peptides and plasma proteins. J. Neurochem., 54: 1882-1888).To separate the vascular and post-vascular compartments, the brain washomogenized in 8 mL cold PBS in a tissue grinder. The homogenate wassupplemented with 9.4 mL cold 40% dextran (70 kDa), and an aliquot ofthe homogenate was taken for radioactivity measurement. The homogenatewas centrifuged at 3200 g at 4C for 10 min in a fixed angle rotor. Thebrain microvasculature quantitatively sediments as the pellet, and thepost-vascular supernatant is a measure of capillary depleted brainparenchyma. The vascular pellet and supernatant were counted for ³Hradioactivity in parallel with the homogenate. The volume ofdistribution (VD) was determined for each of the 3 fractions from theratio of total [¹²⁵I] radioactivity in the brain fraction (DPM/grambrain) divided by the total [¹²⁵I] radioactivity in the 120 min terminalplasma (DPM/uL plasma). The percent of radioactivity in thepost-vascular supernatant that was precipitable with 10% cold TCA wasdetermined Plasma and tissue samples were analyzed for ¹²⁵Iradioactivity with a gamma counter. The VD of the HIRMAb-ASA fusionprotein in brain homogenate at 2 hours after injection is high, 526±23uL/gram, compared to the brain VD of a non-specific human IgG1 isotypecontrol antibody, 20±6 ul/gram (Table 5). The brain VD of the IgG1isotype control antibody represents the brain uptake of a molecule thatis sequestered within the blood volume of brain, and which does notcross the BBB. The high brain VD for the HIRMAb-ASA fusion proteinindicates the fusion protein is either sequestered by the brainvasculature, or has penetrated the BBB and entered brain parenchyma. TheVD of the HIRMAb-ASA fusion protein in the post-vascular supernatant,341±33 uL/gram, is greater than the VD of the HIRMAb-ASA fusion proteinin the vascular pellet of brain, 277±30 uL/gram (Table 5), whichindicates that the majority of the HIRMAb-ASA fusion protein hastraversed the BBB and penetrated the brain parenchyma. The radioactivityin the post-vascular supernatant represents intact HIRMAb-ASA fusionprotein, and not labeled metabolites, as the TCA precipitation of thepost-vascular supernatant radioactivity is 95.2±1.4% (Table 5).

TABLE 5 Capillary depletion analysis for brain uptake of the HIRMAb- ASAfusion protein Molecule Brain fraction VD (μL/g) HIRMAb-ASA fusionprotein Brain homogenate 526 ± 23 Post-vascular supernatant 341 ± 33Vascular pellet 277 ± 30 Human IgG1 isotype control Brain homogenate 20± 6

At 120 minutes after IV injection of the [¹²⁵I]-HIRMAb-ASA fusionprotein, the plasma radioactivity is 42±2% TCA-precipitable, whereas theradioactivity in the post-vascular supernatant of brain is 95±1%TCA-precipitable. This finding means that radioactivity that distributesto brain from blood is the intact HIRMAb-ASA fusion protein, and notradiolabeled metabolites. The results of the capillary depletion methodconfirm the results of the emulsion autoradiography and demonstrated thefusion protein penetrates the BBB at the brain microvasculature and isdelivered to brain cells in the parenchyma of brain.

1. A method for treating an arylsulfatase A (ASA) deficiency in thecentral nervous system of a subject in need thereof, comprisingsystemically administering to the subject a therapeutically effectivedose of a fusion antibody having arylsulfatase A activity, wherein thefusion antibody comprises: (a) a fusion protein comprising the aminoacid sequences of an immunoglobulin heavy chain and an arylsulfatase A;and (b) an immunoglobulin light chain; wherein the fusion antibodycrosses the blood brain barrier (BBB) and the ASA retains at least 20%of its activity, on a molar basis, compared to its activity as aseparate entity.
 2. The method of claim 1, wherein the amino acidsequence of the arylsulfatase A is covalently linked to the carboxyterminus of the amino acid sequence of the immunoglobulin heavy chain.3. The method of claim 1, wherein the fusion antibody ispost-translationally modified by a sulfatase modifying factor type 1(SUMF1).
 4. The method of claim 1, wherein the fusion antibody comprisesformylglycine.
 5. The method of claim 1, wherein the fusion antibodycatalyzes hydrolysis of cerebroside sulfate esters and sulfatidesphingolipids.
 6. (canceled)
 7. The method of claim 1, wherein the ASAand the immunoglobulin each retains at least 20% of its activity, on amolar basis, compared to its activity as a separate entity.
 8. Themethod of claim 1, wherein at least about 100 ug of arylsulfatase Aenzyme are delivered to the brain, normalized per 50 kg body weight. 9.The method of claim 1, wherein the therapeutically effective dosecomprises at least about 10 units/Kg of body weight.
 10. The method ofclaim 1, wherein the arylsulfatase A specific activity of the fusionantibody is at least 10 units/mg of protein.
 11. The method of claim 1,wherein the immunoglobulin heavy chain is an immunoglobulin heavy chainof IgG.
 12. The method of claim 1, wherein the immunoglobulin heavychain comprises a CDR1 corresponding to the amino acid sequence of SEQID NO:1, a CDR2 corresponding to the amino acid sequence of SEQ ID NO:2,or a CDR3 corresponding to the amino acid sequence of SEQ ID NO:3. 13.The method of claim 1, wherein the immunoglobulin light chain is animmunoglobulin light chain of kappa or lambda class.
 14. The method ofclaim 1, wherein the immunoglobulin light chain comprises a CDR1corresponding to the amino acid sequence of SEQ ID NO:4, a CDR2corresponding to the amino acid sequence of SEQ ID NO:5, or a CDR3corresponding to the amino acid sequence of SEQ ID NO:6.
 15. The methodof claim 1, wherein the fusion protein comprising the amino acidsequences of an immunoglobulin heavy chain and an arylsulfatase Acomprises an amino acid sequence that is at least 80% identical to SEQID NO:10.
 16. The method of claim 1, wherein the fusion antibody crossesthe BBB by binding an endogenous BBB receptor-mediated transport system.17. The method of claim 1, wherein the fusion antibody crosses the BBBvia an endogenous BBB receptor selected from the group consisting of theinsulin receptor, transferrin receptor, leptin receptor, lipoproteinreceptor, and the insulin-like growth factor (IGF) receptor.
 18. Themethod of claim 1, wherein the fusion antibody crosses the BBB bybinding an insulin receptor.
 19. The method of claim 1, wherein thesystemic administration is parenteral, intravenous, subcutaneous,intra-muscular, trans-nasal, intra-arterial, transdermal, orrespiratory.
 20. The method of claim 1, wherein the arylsulfatase A(ASA) deficiency in the central nervous system is metachromaticleukodystrophy (MLD). 21.-76. (canceled)